Methods of reducing odor

ABSTRACT

The present invention related to lipase variants having lipase activity and having between 60% to less than 100% sequence identity to a parent lipase or a fragment thereof, wherein the variant comprises one or more substitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/LN, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:10 for position numbering or selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y, L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:2 for position numbering. Further the invention relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; to compositions comprising the variants as well as methods of producing and using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 17/128,344filed on Dec. 21, 2020 (Now Pending) which is a divisional of U.S.application Ser. No. 15/580,853 filed Dec. 8, 2017, now U.S. patent Ser.No. 10/920,203, which is a 35 U.S.C. 371 national application ofPCT/EP2016/065542 filed Jul. 1, 2016, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 15174788.8 filedJul. 1, 2015. The content of each application is fully incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference. The name of the filecontaining the Sequence Listing is SQ.HTML, which was created on Aug. 1,2022 and has 21.9 KB.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the field of cleaning. Morespecifically it relates to methods of reducing odor during lipid stainremoval.

Description of the Related Art

Lipolytic enzymes, i.e., lipases, are used for lipid stain removal.Lipases hydrolyze a broad spectrum of ester bonds some of which resultin release of free fatty acids that generate odor. It is known that whenlipases are included in a wash process an unpleasant malodor may occur.In some instances the problem has been solved by addition of perfumeand/or other fragrance components to mask the odor. There is thus adesire to reduce the odor generated by lipases, in particular duringcleaning.

SUMMARY OF THE INVENTION

The present invention relates to reducing odor generation.

In a first aspect the invention provides lipase variants having lipaseactivity and having between 60% to less than 100% sequence identity to aparent lipase or a fragment thereof, wherein the variant comprises oneor more substitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:10 for position numbering orselected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y,L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y,T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:2 for position numbering.

In a further aspect the invention relates to a method for hydrolyzing alipase substrate comprising: adding to said substrate a lipase variantof a parent lipase, which variant comprises a substitution at one ormore positions corresponding to positions: F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y,L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase, has lipaseactivity, and has at least 60% but less than 100% sequence identity tothe parent lipase or a fragment thereof with lipase activity, in whichmethod odor generation is reduced when compared to the method whereinthe parent lipase is added to the lipase substrate.

In a second aspect the invention relates to a method for lipid stainremoval from a surface comprising contacting said stain with a lipasevariant of a parent lipase, which variant comprises a substitution atone or more positions corresponding to positions: F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y,L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase, has lipaseactivity, and has at least 60% but less than 100% sequence identity tothe parent lipase or a fragment thereof with lipase activity, in whichmethod odor generation is reduced when compared to the method whereinthe parent lipase is added to the lipase substrate.

The present invention also relates to, polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing and using the variants.

Definitions

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may havelipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity(EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-esterhydrolase activity (EC3.1.1.50). In this context a “lipase substrate” isany substrate which can be hydrolyzed by the lipase of the invention.For purposes of the present invention lipase activity (i.e., thehydrolytic activity of the lipase) may be determined with a PnP assayusing substrates with various chain length as described in Example 1. Inone aspect, the variants of the present invention have at least 20%,e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100% of the lipase activity of the parent lipase. In one aspectthe parent lipase is the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12, or afragment thereof with lipase activity.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has lipaseactivity. In one aspect, a fragment contains at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% but less than 100% of thenumber of the amino acids present in the parent lipase. In one aspectthe parent lipase is the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQID NO: 6; SEQ ID NO:8; SEQ ID NO: 10; or SEQ ID NO: 12.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication. Improved property: The term“improved property” means a characteristic associated with a variantthat is improved compared to the parent. Such improved propertiesinclude, but are not limited to reduced odor generation, i.e., odorreduction. Odor generation may be determined as described in Example 1.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 269 of SEQ ID NO: 2; SEQ ID NO: 4; SEQID NO: 6; SEQ ID NO:8; SEQ ID NO: 10; or SEQ ID NO: 12. It is known inthe art that a host cell may produce a mixture of two of more differentmature polypeptides (i.e., with a different C-terminal and/or N-terminalamino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 1 to 807 of SEQ ID NO: 1; SEQ ID NO: 3, SEQ IDNO: 5; SEQ ID NO:7; or SEQ ID NO: 9.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which an alteration is made to produce the enzyme variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof with lipaseactivity. Examples of such parent lipases are those with the amino acidsequences as given in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO:8, SEQ ID NO: 10, or SEQ ID NO: 12.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of apolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving lipase activity. In one aspect, a subsequence contains at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% but lessthan 100% of the number of nucleotides 1 to 807 encoding the parentlipase. In one aspect the parent lipase comprises or consists of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7; or SEQ ID NO: 9.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position. The variantsof the present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% of the lipase activity of the polypeptide of theparent lipase. In one aspect the parent lipase comprises or consists ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, orSEQ ID NO: 12.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, in an embodiment the maturepolypeptide disclosed in SEQ ID NO: 2 is used to determine thecorresponding amino acid residue in another lipase. In anotherembodiment the mature polypeptide disclosed in SEQ ID NO: 10 is used todetermine the corresponding amino acid residue in another lipase.Positions in SEQ ID NO:2 relevant for substitution in the inventioninclude F7, F51, T143, A150, H198, N200, I202, S224, L227, V228, P229,V230, I255, T256, A257, L259, and W260. Positions in SEQ ID NO:10relevant for substitution in the invention include F7, F51, T143, A150,H198, N200, I202, S224, L227, V228, P229, V230, I255, P256, A257, L259,and W260. The amino acid sequence of another lipase is aligned with themature polypeptide disclosed in SEQ ID NO: 2 or SEQ ID NO: 10 as thecase may be, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 2 or SEQ ID NO: 10 is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in another lipasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics26_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 or SEQ ID NO: 10 such that traditional sequence-based comparisonfails to detect their relationship (Lindahl and Elofsson, 2000, J. Mol.Biol. 295: 613-615), other pairwise sequence comparison algorithms canbe used. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg 170Ala”.

DETAILED DESCRIPTION OF THE INVENTION Lipase Variants

The present invention provides lipase variants having lipase activityand having between 60% to less than 100% sequence identity to a parentlipase or a fragment thereof, wherein the variant comprises one or moresubstitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R,F51A/I/L/V/Y, T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P,P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W,A257F/H/I/L/V/Y, L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:10 for position numbering or selected fromH198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V,A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:2 for position numbering.

In an embodiment the lipase variant of the invention comprises thesubstitutions F51A/I/L/V/Y and P256A/K/N/Q/R/S/T/W and one or moresubstitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:10 for position numbering.

In another embodiment the lipase variant of the invention comprises thesubstitutions F51A/I/L/V/Y, preferably F51V, and/orH198A/D/E/F/G/I/L/N/Q/S/T/V/Y, preferably H198S, using SEQ ID NO:2 orSEQ ID NO:10 for position numbering.

In a further embodiment the lipase variant of the invention comprisesthe substitution T256A/K/N/Q/R/S/P/W and one or more substitutionsselected from F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y using SEQ ID NO:2 for position numbering.

In a still further embodiment the lipase variant is a variant of aparent lipase, comprising a substitution at one or more (e.g., several)positions corresponding to positions F7, F51, T143, A150, H198, N200,I202, S224, L227, V228, P229, V230, I255, P256, A257, L259, and W260 ofthe mature polypeptide of the parent lipase, wherein the variant haslipase activity. In one aspect, the lipase variants of a parent lipase,comprise a substitution at one or more (e.g., several) positionscorresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of the parentlipase, wherein the variant has lipase activity. In one aspect, thesequence of the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

In one aspect, the variant has sequence identity of at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, to the amino acid sequence of the parent lipase. Inone aspect, the sequence of the parent lipase is SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

In one aspect, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO:12.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-40, e.g., 1-30, 1-20, 1-10 and 1-5, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 substitutions.

In one aspect, a variant comprises a substitution at one or more (e.g.,several) positions corresponding to positions: 7, 51, 143, 150, 198,200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259, and 260. Inanother aspect, a variant comprises a substitution at two positionscorresponding to any of positions: 7, 51, 143, 150, 198, 200, 202, 224,227, 228, 229, 230, 255, 256, 257, 259, and 260. In another aspect, avariant comprises a substitution at three positions corresponding to anypositions: 7, 51, 143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255,256, 257, 259, and 260. In another aspect, a variant comprises asubstitution at four positions corresponding to any positions: 7, 51,143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259,and 260. In another aspect, a variant comprises a substitution at fivepositions corresponding to any positions: 7, 51, 143, 150, 198, 200,202, 224, 227, 228, 229, 230, 255, 256, 257, 259, and 260. In anotheraspect, a variant comprises a substitution at six positionscorresponding to any positions: 7, 51, 143, 150, 198, 200, 202, 224,227, 228, 229, 230, 255, 256, 257, 259, and 260. In another aspect, avariant comprises a substitution at seven positions corresponding to anypositions: 7, 51, 143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255,256, 257, 259, and 260. In another aspect, a variant comprises asubstitution at eight positions corresponding to any positions: 7, 51,143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259,and 260. In another aspect, a variant comprises a substitution at ninepositions corresponding to any positions: 7, 51, 143, 150, 198, 200,202, 224, 227, 228, 229, 230, 255, 256, 257, 259, and 260. In anotheraspect, a variant comprises a substitution at ten positionscorresponding to any positions: 7, 51, 143, 150, 198, 200, 202, 224,227, 228, 229, 230, 255, 256, 257, 259, and 260. In another aspect, avariant comprises a substitution at eleven positions corresponding toany positions: 7, 51, 143, 150, 198, 200, 202, 224, 227, 228, 229, 230,255, 256, 257, 259, and 260. In another aspect, a variant comprises asubstitution at twelve positions corresponding to any positions: 7, 51,143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259,and 260. In another aspect, a variant comprises a substitution atthirteen positions corresponding to any positions: 7, 51, 143, 150, 198,200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259, and 260. Inanother aspect, a variant comprises a substitution at fourteen positionscorresponding to any positions: 7, 51, 143, 150, 198, 200, 202, 224,227, 228, 229, 230, 255, 256, 257, 259, and 260. In another aspect, avariant comprises a substitution at fifteen positions corresponding toany positions: 7, 51, 143, 150, 198, 200, 202, 224, 227, 228, 229, 230,255, 256, 257, 259, and 260. In another aspect, a variant comprises asubstitution at sixteen positions corresponding to any positions: 7, 51,143, 150, 198, 200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259,and 260. In another aspect, a variant comprises a substitution at eachposition corresponding to positions: 7, 51, 143, 150, 198, 200, 202,224, 227, 228, 229, 230, 255, 256, 257, 259, and 260.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 7. In anotheraspect, the amino acid at a position corresponding to position 7 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Arg,His, or Lys. In another aspect, the variant comprises or consists of thesubstitution F7H, F7K, or F7R of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 51. In anotheraspect, the amino acid at a position corresponding to position 51 issubstituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala,Ile, Leu, Tyr, or Val. In another aspect, the variant comprises orconsists of the substitution F51A, F51I, F51L, F51V, or F51Y of theparent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 143. Inanother aspect, the amino acid at a position corresponding to position143 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Ala, Gly, Ser, or Val. In another aspect, the variant comprises orconsists of the substitution T143A, T143G, T143S, or T143V of the parentlipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 150. Inanother aspect, the amino acid at a position corresponding to position150 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Gly, or Val. In another aspect, the variant comprises or consistsof the substitution A150G, or A150V of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 198. Inanother aspect, the amino acid at a position corresponding to position198 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Phe, Ser, Thr, Tyr, or Val.In another aspect, the variant comprises or consists of the substitutionH198A, H198D, H198E, H198G, H198F, H198, H198Q, H198L, H198N, H198S,H198T, H198V, or H198Y of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 200. Inanother aspect, the amino acid at a position corresponding to position200 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Arg, Gln, His, or Lys. In another aspect, the variant comprises orconsists of the substitution N200H, N200K, N200Q, or N200R of the parentlipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 202. Inanother aspect, the amino acid at a position corresponding to position202 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Gly, Leu, or Val. In another aspect, the variant comprises orconsists of the substitution I202G, I202L, or I202V of the parentlipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 224. Inanother aspect, the amino acid at a position corresponding to position224 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Cys, Phe, His, Ile, Leu, Pro, or Tyr. In another aspect, thevariant comprises or consists of the substitution S224C, S224F, S224H,S224I, S224L, S224P, or S224Y of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 227. Inanother aspect, the amino acid at a position corresponding to position227 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith Asp, Glu, Lys, or Arg. In another aspect, the variant of a parentlipase comprises or consists of the substitution L227D, L227E, L227K, orL227R of the parent lipase.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 228. In another aspect, theamino acid at a position corresponding to position 228 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably with Pro. In anotheraspect, the variant comprises or consists of the substitution V228P ofthe parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 229. Inanother aspect, the amino acid at a position corresponding to position229 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferablywith His, Lys, or Arg. In another aspect, the variant comprises orconsists of the substitution P229H, P229K, or P229R of the parentlipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 230. Inanother aspect, the amino acid at a position corresponding to position230 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withArg, Leu, Lys, or His. In another aspect, the variant comprises orconsists of the substitution V230H, V230K, V230L, or V230R of the parentlipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 255. Inanother aspect, the amino acid at a position corresponding to position255 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withAla, Asn, Gly, Pro, Ser, Thr, Tyr or Val. In another aspect, the variantcomprises or consists of the substitution I255A, I255G, I255N, I255P,I255S, I255T, I255V or I255Y of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 256. Inanother aspect, the amino acid at a position corresponding to position256 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withAla, Arg, Asn, Gln, Lys, Ser, Thr, or Trp. In another aspect, thevariant comprises or consists of the substitution P256A, P256K, P256N,P256Q, P256R, P256S, P256T, or P256W of the parent lipase. In a furtherembodiment the variant substitutions comprises or consists of T256A,T256K, T256N, T256Q, T256R, T256S, T256P, or T256W.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 257. Inanother aspect, the amino acid at a position corresponding to position257 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withHis, Ile, Leu, Phe, Tyr, or Val. In another aspect, the variantcomprises or consists of the substitution A257F, A257H, A257I, A257L,A257V, or A257Y of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 259. Inanother aspect, the amino acid at a position corresponding to position259 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withPhe, or Tyr. In another aspect, the variant comprises or consists of thesubstitution L259F, or L259Y of the parent lipase.

In another aspect, the variant of a parent lipase comprises or consistsof a substitution at a position corresponding to position 260. Inanother aspect, the amino acid at a position corresponding to position260 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val preferably withAsn, Asp, Gln, Glu, His, Ile, Leu, Phe, Ser, Thr, or Tyr. In anotheraspect, the variant comprises or consists of the substitution W260D,W260E, W260F, W260H, W260I, W260L, W260N, W260Q, W260S, W260T, or W260Yof the parent lipase.

In another aspect, the variant comprises or consists of one or more(e.g., several) substitutions selected from the group consisting of:F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/LN, S224C/F/H/I/L/P/Y,L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y,P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y. In one aspect, the variant comprises orconsists of one or more (e.g., several) substitutions selected from thegroup consisting of: F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of SEQ ID NO: 2, orof a polypeptide having at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identity to the mature polypeptide of SEQ ID NO:2 which has lipase activity, and further the variant has reduced odor ascompared to the mature lipase of SEQ ID NO: 2. In one aspect the one ormore (e.g., several) substitutions are one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, or seventeen substitutions. In one aspect, the variantcomprises or consists of one or more (e.g., several) substitutionsselected from the group consisting of: F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y,L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of SEQID NO: 4, or of a polypeptide having at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to the mature polypeptideof SEQ ID NO: 4 which has lipase activity, and further the variant hasreduced odor as compared to the mature lipase of SEQ ID NO: 4. In oneaspect the one or more (e.g., several) substitutions are one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, or seventeen substitutions. In oneaspect, the variant comprises or consists of one or more (e.g., several)substitutions selected from the group consisting of: F7H/K/R,F51A/I/L/V/Y, T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P,P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W,A257F/H/I/L/V/Y, L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y of the maturepolypeptide of SEQ ID NO: 6, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to themature polypeptide of SEQ ID NO: 6 which has lipase activity, andfurther the variant has reduced odor as compared to the mature lipase ofSEQ ID NO: 6. In one aspect the one or more (e.g., several)substitutions are one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeensubstitutions. In one aspect, the variant comprises or consists of oneor more (e.g., several) substitutions selected from the group consistingof: F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of SEQ ID NO: 8, orof a polypeptide having at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identity to the mature polypeptide of SEQ ID NO:8 which has lipase activity, and further the variant has reduced odor ascompared to the mature lipase of SEQ ID NO: 8. In one aspect the one ormore (e.g., several) substitutions are one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, or seventeen substitutions. In one aspect, the variantcomprises or consists of one or more (e.g., several) substitutionsselected from the group consisting of: F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y,L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of SEQID NO: 10, or of a polypeptide having at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to the mature polypeptideof SEQ ID NO: 10 which has lipase activity, and further the variant hasreduced odor as compared to the mature lipase of SEQ ID NO: 10. In oneaspect, the one or more (e.g., several) substitutions are one, two,three, four, five, six, seven, eight, nine, eleven, or twelvesubstitutions. In one aspect the one or more (e.g., several)substitutions are one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeensubstitutions. In one aspect, the variant comprises or consists of oneor more (e.g., several) substitutions selected from the group consistingof: F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/LN, S224C/F/H/I/L/P/Y,L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y,P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of SEQ ID NO: 12, orof a polypeptide having at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% identity to the mature polypeptide of SEQ ID NO:12 which has lipase activity, and further the variant has reduced odoras compared to the mature lipase of SEQ ID NO: 12. In one aspect the oneor more (e.g., several) substitutions are one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or seventeen substitutions.

In one aspect, the the substitutions of the variant of a parent lipasecomprises or consists of the substitutions corresponding to: F7K; F7H;F7R; F51A; F51I; F51L; F51V; F51Y; T143A; T143G; T143S; T143V; A150G;A150V; H198A; H198D; H198E; H198F; H198G; H198I; H198L; H198N; H198Q;H198S; H198T; H198V; H198Y; N200H; N200K; N200Q; N200R; I202G; I202L;I202V; S224C; S224F; S224H; S224I; S224L; S224P; S224Y; L227D; L227E;L227K; L227R; V228P; P229H; P229K; P229R; V230H; V230K; V230L; V230R;I255A; I255G; I255N; I255P; I255S; I255T; I255V; I255Y; P256A; P256K;P256N; P256Q; P256R; P256S; P256T; P256W; T256A, T256K, T256N, T256Q,T256R, T256S, T256P, or T256W; A257F; A257H; A257I; A257L; A257V; A257Y;L259F; L259Y; W260D; W260E; W260F; W260H; W260I; W260L; W260N; W260Q;W260S; W260T; W260Y; N33K F51V K98E; N33K F51V E56K L69R K98E H198I;N33K F51V E56K L69R K98E H198L; N33K F51V E56K L69R K98E H198N; N33KF51V E56K L69R K98E H198S; N33K F51V E56K L69R K98E H198Y; N33K F51VE56K L69R K98E A257V; N33K F51V E56K L69R K98E A257I; N33K F51V E56KL69R K98E I255G; N33K F51V E56K L69R K98E T143A; N33K F51V E56K L69RK98E A150G; N33K F51V E56K L69R K98E V230R; N33K F51V E56K L69R K98EL227D; N33K F51V E56K L69R K98E V228P; N33K F51V E56K L69R K98E S224F;N33K F51V E56K L69R K98E S224I; N33K F51V E56K L69R K98E S224P; V2K N33KF51V H198S; V2K N33K F51V H198Y; V2K N33K F51V H198N; V2K N33K F51VH198I; V2K N33K F51V H198L; V2K N33K F51V A257V; V2K N33K F51V A257I;V2K N33K F51V I255G; V2K N33K F51V T143A; V2K N33K F51V A150G; V2K N33KF51V V230R; V2K N33K F51V L227D; V2K N33K F51V V228P; V2K N33K F51VS224F; V2K N33K F51V S224I; V2K N33K F51V S224P; V2Y N33K F51V H198S;V2Y N33K F51V H198Y; V2Y N33K F51V H198N; V2Y N33K F51V H198I; V2Y N33KF51V H198L; V2Y N33K F51V A257V; V2Y N33K F51V A257I; V2Y N33K F51VI255G; V2Y N33K F51V T143A; V2Y N33K F51V A150G; V2Y N33K F51V V230R;V2Y N33K F51V L227D; V2Y N33K F51V V228P; V2Y N33K F51V S224F; V2Y N33KF51V S224I; V2Y N33K F51V S224P; V2K N33K F51V L69R K98E V176L H198SE210K L227G; V2K N33K F51V L69R K98E V176L H198Y E210K L227G; V2K N33KF51V L69R K98E V176L H198N E210K L227G; V2K N33K F51V L69R K98E V176LH198I E210K L227G; V2K N33K F51V L69R K98E V176L H198L E210K L227G; V2KN33K F51V L69R K98E V176L E210K L227G A257I; V2K N33K F51V L69R K98EV176L E210K L227G A257V; V2K N33K F51V L69R K98E V176L E210K L227GI255G; V2K N33K F51V L69R K98E T143A V176L E210K L227G; V2K N33K F51VL69R K98E A150G V176L E210K L227G; V2K N33K F51V L69R K98E V176L E210KL227G V230R; V2K N33K F51V L69R K98E V176L E210K L227G V228P; V2K N33KF51V L69R K98E V176L E210K S224F L227G; V2K N33K F51V L69R K98E V176LE210K S224I L227G; V2K N33K F51V L69R K98E V176L E210K S224P L227G; V2KN33K F51V L69R K98E V176L E210K L227D; V2Y N33K F51V L69R K98E V176LH198S E210K L227G; V2Y N33K F51V L69R K98E V176L H198Y E210K L227G; V2YN33K F51V L69R K98E V176L H198N E210K L227G; V2Y N33K F51V L69R K98EV176L H198I E210K L227G; V2Y N33K F51V L69R K98E V176L H198L E210KL227G; V2Y N33K F51V L69R K98E V176L E210K L227G A257I; V2Y N33K F51VL69R K98E V176L E210K L227G A257V; V2Y N33K F51V L69R K98E V176L E210KL227G I255G; V2Y N33K F51V L69R K98E T143A V176L E210K L227G; V2Y N33KF51V L69R K98E A150G V176L E210K L227G; V2Y N33K F51V L69R K98E V176LE210K L227G V230R; V2Y N33K F51V L69R K98E V176L E210K L227G V228P; V2YN33K F51V L69R K98E V176L E210K S224F L227G; V2Y N33K F51V L69R K98EV176L E210K S224I L227G; V2Y N33K F51V L69R K98E V176L E210K S224PL227G; V2Y N33K F51V L69R K98E V176L E210K L227D; N33K F51V S54T E56KK98I H198S; N33K F51V S54T E56K K98I H198Y; N33K F51V S54T E56K K98IH198N; N33K F51V S54T E56K K98I H198I; N33K F51V S54T E56K K98I H198L;N33K F51V S54T E56K K98I A257V; N33K F51V S54T E56K K98I A257I; N33KF51V S54T E56K K98I I255G; N33K F51V S54T E56K K98I T143A; N33K F51VS54T E56K K98I A150G; N33K F51V S54T E56K K98I V230R; N33K F51V S54TE56K K98I L227D; N33K F51V S54T E56K K98I V228P; N33K F51V S54T E56KK98I S224F; N33K F51V S54T E56K K98I S224I; N33K F51V S54T E56K K98IS224P; E1C V2K N33K F51V H198S R233C; E1C V2K N33K F51V H198Y R233C; E1CV2K N33K F51V H198N R233C; E1C V2K N33K F51V H198I R233C; E1C V2K N33KF51V H198L R233C; E1C V2K N33K F51V R233C A257I; E1C V2K N33K F51V R233CA257V; E1C V2K N33K F51V R233C I255G; E1C V2K N33K F51V T143A R233C; E1CV2K N33K F51V A150G R233C; E1C V2K N33K F51V V230R R233C; E1C V2K N33KF51V L227D R233C; E1C V2K N33K F51V V228P R233C; E1C V2K N33K F51V S224FR233C; E1C V2K N33K F51V S224I R233C; E1C V2K N33K F51V S224P R233C; E1CV2Y N33K F51V H198S R233C; E1C V2Y N33K F51V H198Y R233C; E1C V2Y N33KF51V H198N R233C; E1C V2Y N33K F51V H198I R233C; E1C V2Y N33K F51V H198LR233C; E1C V2Y N33K F51V R233C A257I; E1C V2Y N33K F51V R233C A257V; E1CV2Y N33K F51V R233C I255G; E1C V2Y N33K F51V T143A R233C; E1C V2Y N33KF51V A150G R233C; E1C V2Y N33K F51V V230R R233C; E1C V2Y N33K F51V L227DR233C; E1C V2Y N33K F51V V228P R233C; E1C V2Y N33K F51V S224F R233C; E1CV2Y N33K F51V S224I R233C; E1C V2Y N33K F51V S224P R233C; N33K F51V K98EN101D H198S; N33K F51V K98E N101D H198S E210R Y220F A257V; N33K F51VK98E N101D H198S E210K Y220F A257V; N33K F51V K98E N101D H198S E210KY220F A257F; N33K F51V K98E N101D H198S E210K Y220F A257H; N33K F51VK98E N101D H198D E210K Y220F A257V; N33K F51V K98E N101D H198S E210KY220F V228P; N33K F51V K98E N101D H198S E210K S224P; N33K F51V K98EN101D G161N H198S E210K Y220F A257V; N33K F51L E56K L69R K98E H198I;N33K F51L E56K L69R K98E H198L; N33K F51L E56K L69R K98E H198N; N33KF51L E56K L69R K98E H198S; N33K F51L E56K L69R K98E H198Y; N33K F51LE56K L69R K98E A257V; N33K F51L E56K L69R K98E A257I; N33K F51L E56KL69R K98E I255G; N33K F51L E56K L69R K98E T143A; N33K F51L E56K L69RK98E A150G; N33K F51L E56K L69R K98E V230R; N33K F51L E56K L69R K98EL227D; N33K F51L E56K L69R K98E L228P; N33K F51L E56K L69R K98E S224F;N33K F51L E56K L69R K98E S224I; N33K F51L E56K L69R K98E S224P; V2K N33KF51L H198S; V2K N33K F51L H198Y; V2K N33K F51L H198N; V2K N33K F51LH198I; V2K N33K F51L H198L; V2K N33K F51L A257V; V2K N33K F51L A257I;V2K N33K F51L I255G; V2K N33K F51L T143A; V2K N33K F51L A150G; V2K N33KF51L V230R; V2K N33K F51L L227D; V2K N33K F51L V228P; V2K N33K F51LS224F; V2K N33K F51L S224I; V2K N33K F51L S224P; V2Y N33K F51L H198S;V2Y N33K F51L H198Y; V2Y N33K F51L H198N; V2Y N33K F51L H198I; V2Y N33KF51L H198L; V2Y N33K F51L A257V; V2Y N33K F51L A257I; V2Y N33K F51LI255G; V2Y N33K F51L T143A; V2Y N33K F51L A150G; V2Y N33K F51L V230R;V2Y N33K F51L L227D; V2Y N33K F51L V228P; V2Y N33K F51L S224F; V2Y N33KF51L S224I; V2Y N33K F51L S224P; V2K N33K F51L L69R K98E V176L H198SE210K L227G; V2K N33K F51L L69R K98E V176L H198Y E210K L227G; V2K N33KF51L L69R K98E V176L H198N E210K L227G; V2K N33K F51L L69R K98E V176LH198I E210K L227G; V2K N33K F51L L69R K98E V176L H198L E210K L227G; V2KN33K F51L L69R K98E V176L E210K L227G A257I; V2K N33K F51L L69R K98EV176L E210K L227G A257V; V2K N33K F51L L69R K98E V176L E210K L227GI255G; V2K N33K F51L L69R K98E T143A V176L E210K L227G; V2K N33K F51LL69R K98E A150G V176L E210K L227G; V2K N33K F51L L69R K98E V176L E210KL227G V230R; V2K N33K F51L L69R K98E V176L E210K L227G V228P; V2K N33KF51L L69R K98E V176L E210K S224F L227G; V2K N33K F51L L69R K98E V176LE210K S224I L227G; V2K N33K F51L L69R K98E V176L E210K S224P L227G; V2KN33K F51L L69R K98E V176L E210K L227D; V2Y N33K F51L L69R K98E V176LH198S E210K L227G; V2Y N33K F51L L69R K98E V176L H198Y E210K L227G; V2YN33K F51L L69R K98E V176L H198N E210K L227G; V2Y N33K F51L L69R K98EV176L H198I E210K L227G; V2Y N33K F51L L69R K98E V176L H198L E210KL227G; V2Y N33K F51L L69R K98E V176L E210K L227G A257I; V2Y N33K F51LL69R K98E V176L E210K L227G A257V; V2Y N33K F51L L69R K98E V176L E210KL227G I255G; V2Y N33K F51L L69R K98E T143A V176L E210K L227G; V2Y N33KF51L L69R K98E A150G V176L E210K L227G; V2Y N33K F51L L69R K98E V176LE210K L227G V230R; V2Y N33K F51L L69R K98E V176L E210K L227G V228P; V2YN33K F51L L69R K98E V176L E210K S224F L227G; V2Y N33K F51L L69R K98EV176L E210K S224I L227G; V2Y N33K F51L L69R K98E V176L E210K S224PL227G; V2Y N33K F51L L69R K98E V176L E210K L227D; N33K F51L S54T E56KK98I H198S; N33K F51L S54T E56K K98I H198Y; N33K F51L S54T E56K K98IH198N; N33K F51L S54T E56K K98I H198I; N33K F51L S54T E56K K98I H198L;N33K F51L S54T E56K K98I A257V; N33K F51L S54T E56K K98I A257I; N33KF51L S54T E56K K98I I255G; N33K F51L S54T E56K K98I T143A; N33K F51LS54T E56K K98I A150G; N33K F51L S54T E56K K98I V230R; N33K F51L S54TE56K K98I L227D; N33K F51L S54T E56K K98I V228P; N33K F51L S54T E56KK98I S224F; N33K F51L S54T E56K K98I S224I; N33K F51L S54T E56K K98IS224P; E1C V2K N33K F51L H198S R233C; E1C V2K N33K F51L H198Y R233C; E1CV2K N33K F51L H198N R233C; E1C V2K N33K F51L H198I R233C; E1C V2K N33KF51L H198L R233C; E1C V2K N33K F51L R233C A257I; E1C V2K N33K F51L R233CA257V; E1C V2K N33K F51L R233C I255G; E1C V2K N33K F51L T143A R233C; E1CV2K N33K F51L A150G R233C; E1C V2K N33K F51L V230R R233C; E1C V2K N33KF51L L227D R233C; E1C V2K N33K F51L V228P R233C; E1C V2K N33K F51L S224FR233C; E1C V2K N33K F51L S224I R233C; E1C V2K N33K F51L S224P R233C; E1CV2Y N33K F51L H198S R233C; E1C V2Y N33K F51L H198Y R233C; E1C V2Y N33KF51L H198N R233C; E1C V2Y N33K F51L H198I R233C; E1C V2Y N33K F51L H198LR233C; E1C V2Y N33K F51L R233C A257I; E1C V2Y N33K F51L R233C A257V; E1CV2Y N33K F51L R233C I255G; E1C V2Y N33K F51L T143A R233C; E1C V2Y N33KF51L A150G R233C; E1C V2Y N33K F51L V230R R233C; E1C V2Y N33K F51L L227DR233C; E1C V2Y N33K F51L V228P R233C; E1C V2Y N33K F51L S224F R233C; E1CV2Y N33K F51L S224I R233C; E1C V2Y N33K F51L S224P R233C; N33K F51L K98EN101D H198S; N33K F51L K98E N101D H198S E210R Y220F A257V; N33K F51LK98E N101D H198S E210K Y220F A257V; N33K F51L K98E N101D H198S E210KY220F A257F; N33K F51L K98E N101D H198S E210K Y220F A257H; N33K F51LK98E N101D H198D E210K Y220F A257V; N33K F51L K98E N101D H198SE210KY220F V228P; N33K F51L K98E N101D H198S E210K S224P; N33K F51L K98EN101D G161N H198S E210K Y220F A257V; N33K F51I E56K L69R K98E H198I;N33K F51I E56K L69R K98E H198L; N33K F51I E56K L69R K98E H198N; N33KF51I E56K L69R K98E H198S; N33K F51I E56K L69R K98E H198Y; N33K F51IE56K L69R K98E A257V; N33K F51I E56K L69R K98E A257I; N33K F51I E56KL69R K98E I255G; N33K F51I E56K L69R K98E T143A; N33K F51I E56K L69RK98E A150G; N33K F51I E56K L69R K98E V230R; N33K F51I E56K L69R K98EL227D; N33K F51I E56K L69R K98E V228P; N33K F51I E56K L69R K98E S224F;N33K F51I E56K L69R K98E S224I; N33K F51I E56K L69R K98E S224P; V2K N33KF51I H198S; V2K N33K F51I H198Y; V2K N33K F51I H198N; V2K N33K F51IH198I; V2K N33K F51I H198L; V2K N33K F51I A257V; V2K N33K F51I A257I;V2K N33K F51I I255G; V2K N33K F51I T143A; V2K N33K F51I A150G; V2K N33KF51I V230R; V2K N33K F51I L227D; V2K N33K F51I V228P; V2K N33K F51IS224F; V2K N33K F51I S224I; V2K N33K F51I S224P; V2Y N33K F51I H198S;V2Y N33K F51I H198Y; V2Y N33K F51I H198N; V2Y N33K F51I H198I; V2Y N33KF51I H198L; V2Y N33K F51I A257V; V2Y N33K F51I A257I; V2Y N33K F51II255G; V2Y N33K F51I T143A; V2Y N33K F51I A150G V2Y N33K F51I V230R; V2YN33K F51I L227D; V2Y N33K F51I V228P; V2Y N33K F51I S224F; V2Y N33K F51IS224I; V2Y N33K F51I S224P; V2K N33K F51I L69R K98E V176L H198S E210KL227G; V2K N33K F51I L69R K98E V176L H198Y E210K L227G; V2K N33K F51IL69R K98E V176L H198N E210K L227G; V2K N33K F51I L69R K98E V176L H198IE210K L227G; V2K N33K F51I L69R K98E V176L H198L E210K L227G; V2K N33KF51I L69R K98E V176L E210K L227G A257I; V2K N33K F51I L69R K98E V176LE210K L227G A257V; V2K N33K F51I L69R K98E V176L E210K L227G I255G; V2KN33K F51I L69R K98E T143A V176L E210K L227G; V2K N33K F51I L69R K98EA150G V176L E210K L227G; V2K N33K F51I L69R K98E V176L E210K L227GV230R; V2K N33K F51I L69R K98E V176L E210K L227G V228P; V2K N33K F51IL69R K98E V176L E210K S224F L227G; V2K N33K F51I L69R K98E V176L E210KS224I L227G; V2K N33K F51I L69R K98E V176L E210K S224P L227G; V2K N33KF51I L69R K98E V176L E210K L227D; V2Y N33K F51I L69R K98E V176L H198SE210K L227G; V2Y N33K F51I L69R K98E V176L H198Y E210K L227G; V2Y N33KF51I L69R K98E V176L H198N E210K L227G; V2Y N33K F51I L69R K98E V176LH198I E210K L227G; V2Y N33K F51I L69R K98E V176L H198L E210K L227G; V2YN33K F51I L69R K98E V176L E210K L227G A257I; V2Y N33K F51I L69R K98EV176L E210K L227G A257V; V2Y N33K F51I L69R K98E V176L E210K L227GI255G; V2Y N33K F51I L69R K98E T143A V176L E210K L227G; V2Y N33K F51IL69R K98E A150G V176L E210K L227G; V2Y N33K F51I L69R K98E V176L E210KL227G V230R; V2Y N33K F51I L69R K98E V176L E210K L227G V228P; V2Y N33KF51I L69R K98E V176L E210K S224F L227G; V2Y N33K F51I L69R K98E V176LE210K S224I L227G; V2Y N33K F51I L69R K98E V176L E210K S224P L227G; V2YN33K F51I L69R K98E V176L E210K L227D; N33K F51I S54T E56K K98I H198S;N33K F51I S54T E56K K98I H198Y; N33K F51I S54T E56K K98I H198N; N33KF51I S54T E56K K98I H198I; N33K F51I S54T E56K K98I H198L; N33K F51IS54T E56K K98I A257V; N33K F51I S54T E56K K98I A257I; N33K F51I S54TE56K K98I I255G; N33K F51I S54T E56K K98I T143A; N33K F51I S54T E56KK98I A150G; N33K F51I S54T E56K K98I V230R; N33K F51I S54T E56K K98IL227D; N33K F51I S54T E56K K98I V228P; N33K F51I S54T E56K K98I S224F;N33K F51I S54T E56K K98I S224I; N33K F51I S54T E56K K98I S224P; E1C V2KN33K F51I H198S R233C; E1C V2K N33K F51I H198Y R233C; E1C V2K N33K F51IH198N R233C; E1C V2K N33K F51I H198I R233C; E1C V2K N33K F51I H198LR233C; E1C V2K N33K F51I R233C A257I; E1C V2K N33K F51I R233C A257V; E1CV2K N33K F51I R233C I255G; E1C V2K N33K F51I T143A R233C; E1C V2K N33KF51I A150G R233C; E1C V2K N33K F51I V230R R233C; E1C V2K N33K F51I L227DR233C; E1C V2K N33K F51I V228P R233C; E1C V2K N33K F51I S224F R233C; E1CV2K N33K F51I S224I R233C; E1C V2K N33K F51I S224P R233C; E1C V2Y N33KF51I H198S R233C; E1C V2Y N33K F51I H198Y R233C; E1C V2Y N33K F51I H198NR233C; E1C V2Y N33K F51I H198I R233C; E1C V2Y N33K F51I H198L R233C; E1CV2Y N33K F51I R233C A257I; E1C V2Y N33K F51I R233C A257V; E1C V2Y N33KF51I R233C I255G; E1C V2Y N33K F51I T143A R233C; E1C V2Y N33K F51I A150GR233C; E1C V2Y N33K F51I V230R R233C; E1C V2Y N33K F51I L227D R233C; E1CV2Y N33K F51I V228P R233C; E1C V2Y N33K F51I S224F R233C; E1C V2Y N33KF51I S224I R233C; E1C V2Y N33K F51I S224P R233C; N33K F51I K98E N101DH198S; N33K F51I K98E N101D H198S E210R Y220F A257V; N33K F51I K98EN101D H198S E210K Y220F A257V; N33K F51I K98E N101D H198S E210K Y220FA257F; N33K F51I K98E N101D H198S E210K Y220F A257H; N33K F51I K98EN101D H198D E210K Y220F A257V; N33K F51I K98E N101D H198S E210K Y220FV228P; N33K F51I K98E N101D H198S E210K S224P; N33K F51I K98E N101DG161N H198S E210K Y220F A257V; G91T P256T; N33Q G91T P256T; L259F W260H;V230L L259F W260H; and I255T A257V of the mature polypeptide of theparent lipase. In one aspect the parent lipase is SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

For example, the variants may comprise a substitution at a positioncorresponding to any of positions; 1, 2, 4, 27, 33, 38, 54, 56, 57, 58,60, 69, 83, 86, 91, 94, 97, 98, 99, 101, 111, 163, 176, 210, 216, 220,225, 227, 231, 233, 249, 254, 255, 256, 263, 264, 265, 266, 267, and 269of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, or SEQ ID NO: 12. In one aspect the variants further comprising oneor more (e.g., several) substitutions corresponding to any of positionsselected from: 1, 2, 4, 27, 33, 38, 54, 56, 57, 58, 60, 69, 83, 86, 91,94, 97, 98, 99, 101, 111, 163, 176, 210, 216, 220, 225, 227, 231, 233,249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO:12. In one aspect the variants further comprising one or more (e.g.,several) substitutions corresponding to any of positions selected from:E1C, V2K/Y, Q4V, D27R, N33K/Q, G38A, S54T, E56K, D57G, 558A, V60S, L69R,S83T, 186V, G91A/N/Q/T, N94K/R, D96E/G/L/W, L97M, K98E/I, E99K, N101D,D111A, G163K, V176L, E210K/Q/R, S216P, Y220F, G225R, L227G, T231R,N233C/R, Q249R, D254S, P256V, G263Q, L264A, I265T, G266D, T267A, andL269N of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 10, or SEQ ID NO: 12, preferably using SEQ ID NO:10 or SEQ ID NO: 2for numbering.

In some embodiments, lipase variants of the invention provide both areduced odor generation as well as an increased stability of thevariant, e.g., during storage, such as storage in a hostile environmentsuch as in a detergent composition. In this context the term “hostileenvironment”, means an environment which actively interacts negativelywith the lipase variant and reduces its lipase activity eitherreversibly or irreversibly. In this embodiment the invention provideslipase variants, wherein the variant comprises the substitution F51V andone or more substitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,F7H/K/R, T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/W or T256A/K/N/Q/R/S/P/W,A257F/H/I/L/V/Y, L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y, exhibiting botha reduced odor generation upon hydrolyzing a lipase substrate and animproved stability compared to the parent lipase.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, e.g., de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identity of essential amino acids can also beinferred from an alignment with a related polypeptide.

The variants may consist or contain at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the number of amino acids of theparent lipase. In one aspect, the variant has reduced odor as comparedto the parent enzyme. In one aspect, the parent enzyme comprises orconsists of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQID NO: 10, or SEQ ID NO: 12; or a fragment thereof with lipase activity.Odor reduction may be determined as described in the examples. In anembodiment the parent lipase is SEQ ID NO: 2 or SEQ ID NO: 10 XX

Parent Lipase

The parent lipase may be (a) a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12; (b) a polypeptide encodedby a polynucleotide that hybridizes under low stringency conditions with(i) the polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO:7; SEQ ID NO: 9; or SEQ ID NO: 11, (ii) thefull-length complement of (i); or (c) a polypeptide encoded by apolynucleotide having at least 60% sequence identity to the polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO:7; SEQ ID NO: 9; or SEQ ID NO: 11.

In one aspect, the parent has a sequence identity to the polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, orSEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have lipaseactivity. In one aspect, the amino acid sequence of the parent differsby up to 40 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 from the polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ IDNO: 12.

In one aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQID NO: 10, or SEQ ID NO: 12.

In one aspect, the parent is a fragment of the polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO:12, containing at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% of the number of amino acids of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12.

In one aspect, the parent is an allelic variant of the polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, orSEQ ID NO: 12.

In one aspect, the parent is encoded by a polynucleotide that hybridizesunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) thepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5SEQ ID NO:7; or SEQ ID NO: 9, (ii) the full-length complement of (i)(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO:7; SEQ ID NO: 9; or SEQ ID NO: 11, or a subsequence thereof, as wellas the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO:8, SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, or SEQ ID NO: 11, or a subsequence thereof, the carrier material isused in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, or SEQ ID NO: 11; (ii) the polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ IDNO: 11; (iii) the full-length complement thereof; or (iv) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,or SEQ ID NO: 11. In one aspect, the nucleic acid probe is at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the number ofnucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, or SEQ ID NO: 11. In one aspect, the nucleic acid probe isa polynucleotide that encodes the polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12; themature polypeptide thereof; or a fragment thereof. In one aspect, thenucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

In one aspect, the parent is encoded by a polynucleotide having asequence identity to the polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial lipase. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis lipase.

In one aspect, the parent is a Streptococcus equisimilis, Streptococcuspyogenes, Streptococcus uberis, or Streptococcus equi subsp.Zooepidemicus lipase. In one aspect, the parent is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans lipase.

In one aspect, the parent is a Thermobifida alba or Thermobifida fusca(formerly known as Thermomonaspora fusca) lipase.

The parent may be a fungal lipase. For example, the parent may be ayeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipasesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria lipase.

In one aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis lipase.

In one aspect, the parent is an Acremonium cellulolyticus, Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusfumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Chrysosporium inops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporiumtropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,lrpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

In one aspect, the parent is a Thermomyces lanuginosus lipase, e.g., thelipase of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ IDNO: 10, or SEQ ID NO: 12, or a fragment thereof with lipase activity.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a variant ofa parent lipase having lipase activity, comprising: (a) introducing intothe parent lipase a substitution at one or more (e.g., several)positions corresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V,A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase, wherein the variant haslipase activity; and (b) recovering the variant. In one aspect theparent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8,SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof with lipaseactivity.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US 2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase,Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The invention further furnish methods for obtaining a lipase variant ofthe invention, comprising introducing prescribed substitutions into aparent lipase and recovering the variant. The invention further furnishmethods for obtaining a lipase variant of a parent lipase, comprisingintroducing into the parent lipase a substitution at one or morepositions corresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V,A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/LN,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase which parent lipasecomprises or consists of the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, or afragment thereof with lipase activity, wherein the variant has lipaseactivity; and recovering the variant.

In an embodiment the invention provides methods for producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant such as thosedescribed in the examples.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

Compositions comprising the polypeptide of the present inventions arecontemplated. In certain aspects the present invention relates to adetergent composition comprising a lipase variant of the invention suchas a lipase variant of a parent lipase, wherein the variant has lipaseactivity, comprises or constitutes a substitution at one or morepositions corresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V,A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/LN,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of the parentlipase, wherein the variant has lipase activity and has at least 60% butless than 100% sequence identity to the mature polypeptide of the parentlipase or a fragment thereof with lipase activity. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO:8;SEQ ID NO: 10; or SEQ ID NO: 12.

In one aspect the invention relates to compositions comprising lipasevariants further comprising one or more (e.g., several) substitutionscorresponding to any of positions selected from: 1, 2, 4, 27, 33, 38,54, 56, 57, 58, 60, 69, 83, 86, 91, 94, 97, 98, 99, 101, 111, 163, 176,210, 216, 220, 225, 227, 231, 233, 249, 254, 255, 256, 263, 264, 265,266, 267, and 269 of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ IDNO:8; SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect the inventionrelates to compositions comprising lipase variants further comprisingone or more (e.g., several) substitutions corresponding to any ofpositions selected from: E1C, V2K/Y, Q4V, D27R, N33K/Q, G38A, S54T,E56K, D57G, S58A, V60S, L69R, S83T, 186V, G91A/N/Q/T, N94K/R,D96E/G/L/W, L97M, K98E/I, E99K, N101D, D111A, G163K, V176L, E210K/Q/R,S216P, Y220F, G225R, L227G, T231R, N233C/R, Q249R, D254S, P256V, G263Q,L264A, I265T, G266D, T267A, and L269N of SEQ ID NO: 2; SEQ ID NO: 4; SEQID NO: 6; SEQ ID NO:8; SEQ ID NO: 10; or SEQ ID NO: 12.

In one aspect said variant has reduced odor generation in comparisonwith the parent lipase.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain aspects of the invention, e.g., toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Unless otherwise indicated the amounts in percentage is by weight of thecomposition (wt. %). Suitable component materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, enzymes, and enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, hueing dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents and/or pigments. In addition to the disclosure below,suitable examples of such other components and levels of use are foundin U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348 herebyincorporated by reference.

Thus, in certain aspects the invention do not contain one or more of thefollowing adjuncts materials: surfactants, soaps, builders, chelatingagents, dye transfer inhibiting agents, dispersants, additional enzymes,enzyme stabilizers, catalytic materials, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, perfume delivery systems,structure elasticizing agents, fabric softeners, carriers, hydrotropes,processing aids, solvents and/or pigments. However, when one or morecomponents are present, such one or more components may be present asdetailed below:

Surfactants—The compositions according to the present invention maycomprise a surfactant or surfactant system wherein the surfactant can beselected from nonionic surfactants, anionic surfactants, cationicsurfactants, ampholytic surfactants, zwitterionic surfactants,semi-polar nonionic surfactants and mixtures thereof. When present,surfactant is typically present at a level of from 0.1 to 60 wt. %, from0.2 to 40 wt. %, from 0.5 to 30 wt. %, from 1 to 50 wt. %, from 1 to 40wt. %, from 1 to 30 wt. %, from 1 to 20 wt. %, from 3 to 10 wt. %, from3 to 5 wt. %, from 5 to 40 wt. %, from 5 to 30 wt. %, from 5 to 15 wt.%, from 3 to 20 wt. %, from 3 to 10 wt. %, from 8 to 12 wt. %, from 10to 12 wt. %, from 20 to 25 wt. % or from 25-60 wt. %.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in one aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g., a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Non-limiting examples of anionic surfactants include sulfates andsulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomersof LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof. Anionic surfactants can be added as correspondingacids or as salts, or can be used as derivatives with ethanolamines, exmonoethanolamine-linear alkylbenzensulfonate (MEA-LAS).

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄-C₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g., a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includeLutensol®.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), as well as products available underthe trade names SPAN and TWEEN, and combinations thereof.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₈₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g., chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Non-limiting examples of cationic surfactants includealkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, ester quats, andcombinations thereof.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines such as alkyldimethylbetaines, sulfobetaines, or combinationsthereof. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g., part ofthe anionic surfactant mix may be neutralized with sodium or potassiumand part of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide.

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

In one aspect a surfactant system may coprise a mixture of isoprenoidsurfactants represented by formula A and formula B:

where Y is CH₂ or null, and Z may be chosen such that the resultingsurfactant is selected from the following surfactants: an alkylcarboxylate surfactant, an alkyl polyalkoxy surfactant, an alkyl anionicpolyalkoxy sulfate surfactant, an alkyl glycerol ester sulfonatesurfactant, an alkyl dimethyl amine oxide surfactant, an alkylpolyhydroxy based surfactant, an alkyl phosphate ester surfactant, analkyl glycerol sulfonate surfactant, an alkyl polygluconate surfactant,an alkyl polyphosphate ester surfactant, an alkyl phosphonatesurfactant, an alkyl polyglycoside surfactant, an alkyl monoglycosidesurfactant, an alkyl diglycoside surfactant, an alkyl sulfosuccinatesurfactant, an alkyl disulfate surfactant, an alkyl disulfonatesurfactant, an alkyl sulfosuccinamate surfactant, an alkyl glucamidesurfactant, an alkyl taurinate surfactant, an alkyl sarcosinatesurfactant, an alkyl glycinate surfactant, an alkyl isethionatesurfactant, an alkyl dialkanolamide surfactant, an alkylmonoalkanolamide surfactant, an alkyl monoalkanolamide sulfatesurfactant, an alkyl diglycolamide surfactant, an alkyl diglycolamidesulfate surfactant, an alkyl glycerol ester surfactant, an alkylglycerol ester sulfate surfactant, an alkyl glycerol ether surfactant,an alkyl glycerol ether sulfate surfactant, alkyl methyl ester sulfonatesurfactant, an alkyl polyglycerol ether surfactant, an alkylpolyglycerol ether sulfate surfactant, an alkyl sorbitan estersurfactant, an alkyl ammonioalkanesulfonate surfactant, an alkylamidopropyl betaine surfactant, an alkyl allylated quat basedsurfactant, an alkyl monohydroxyalkyl-di-alkylated quat basedsurfactant, an alkyl di-hydroxyalkyl monoalkyl quat based surfactant, analkylated quat surfactant, an alkyl trimethylammonium quat surfactant,an alkyl polyhydroxalkyl oxypropyl quat based surfactant, an alkylglycerol ester quat surfactant, an alkyl glycol amine quat surfactant,an alkyl monomethyl dihydroxyethyl quaternary ammonium surfactant, analkyl dimethyl monohydroxyethyl quaternary ammonium surfactant, an alkyltrimethylammonium surfactant, an alkyl imidazoline-based surfactant, analken-2-yl-succinate surfactant, an alkyl a-sulfonated carboxylic acidsurfactant, an alkyl a-sulfonated carboxylic acid alkyl estersurfactant, an alpha olefin sulfonate surfactant, an alkyl phenolethoxylate surfactant, an alkyl benzenesulfonate surfactant, an alkylsulfobetaine surfactant, an alkyl hydroxysulfobetaine surfactant, analkyl ammoniocarboxylate betaine surfactant, an alkyl sucrose estersurfactant, an alkyl alkanolamide surfactant, an alkyldi(polyoxyethylene) monoalkyl ammonium surfactant, an alkylmono(polyoxyethylene) dialkyl ammonium surfactant, an alkyl benzyldimethylammonium surfactant, an alkyl aminopropionate surfactant, analkyl amidopropyl dimethylamine surfactant, or a mixture thereof; and ifZ is a charged moiety, Z is charge-balanced by a suitable metal ororganic counter ion. Suitable counter ions include a metal counter ion,an amine, or an alkanolamine, e.g., C1-C6 alkanolammonium. Morespecifically, suitable counter ions include Na+, Ca+, Li+, K+, Mg+,e.g., monoethanolamine (MEA), diethanolamine (DEA), triethanolamine(TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine,dimethylethanolamine, monoisopropanolamine, triisopropanolamine,1-amino-3-propanol, or mixtures thereof. In one aspect, the compositionscontain from 5% to 97% of one or more non-isoprenoid surfactants; andone or more adjunct cleaning additives; wherein the weight ratio ofsurfactant of formula A to surfactant of formula B is from 50:50 to95:5.

Soap—The compositions herein may contain soap. Without being limited bytheory, it may be desirable to include soap as it acts in part as asurfactant and in part as a builder and may be useful for suppression offoam and may furthermore interact favorably with the various cationiccompounds of the composition to enhance softness on textile fabricstreaded with the inventive compositions. Any soap known in the art foruse in laundry detergents may be utilized. In one aspect, thecompositions contain from 0 to 20 wt. %, from 0.5 to 20 wt. %, from 4 to10 wt. %, or from 4 to 7 wt. % of soap. In one aspect, the compositionscontain from 0 to 2 wt. %, from 0 to 1.5 wt. %, from 0 to 1 wt. %, orfrom 0 to 0.5 wt. % of soap.

Examples of soap useful herein include oleic acid soaps, palmitic acidsoaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soapsare in the form of mixtures of fatty acid soaps having different chainlengths and degrees of substitution. One such mixture is topped palmkernel fatty acid.

In one aspect, the soap is selected from free fatty acid. Suitable fattyacids are saturated and/or unsaturated and can be obtained from naturalsources such a plant or animal esters (e.g., palm kernel oil, palm oil,coconut oil, babassu oil, safflower oil, tall oil, castor oil, tallowand fish oils, grease, and mixtures thereof), or synthetically prepared(e.g., via the oxidation of petroleum or by hydrogenation of carbonmonoxide via the Fisher Tropsch process).

Examples of suitable saturated fatty acids for use in the compositionsof this invention include captic, lauric, myristic, palmitic, stearic,arachidic and behenic acid. Suitable unsaturated fatty acid speciesinclude: palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.Examples of preferred fatty acids are saturated Cn fatty acid, saturatedCi₂-Ci₄ fatty acids, and saturated or unsaturated Cn to Ci₈ fatty acids,and mixtures thereof.

When present, the weight ratio of fabric softening cationic cosurfactantto fatty acid is preferably from about 1:3 to about 3:1, more preferablyfrom about 1:1.5 to about 1.5:1, most preferably about 1:1.

Levels of soap and of nonsoap anionic surfactants herein are percentagesby weight of the detergent composition, specified on an acid form basis.However, as is commonly understood in the art, anionic surfactants andsoaps are in practice neutralized using sodium, potassium oralkanolammonium bases, such as sodium hydroxide or monoethanolamine.

Hydrotropes—The compositions of the present invention may comprise oneor more hydrotropes. A hydrotrope is a compound that solubiliseshydrophobic compounds in aqueous solutions (or oppositely, polarsubstances in a non-polar environment). Typically, hydrotropes have bothhydrophilic and a hydrophobic character (so-called amphiphilicproperties as known from surfactants); however the molecular structureof hydrotropes generally do not favor spontaneous self-aggregation, see,e.g., review by Hodgdon and Kaler, 2007, Current Opinion in Colloid &Interface Science 12: 121-128. Hydrotropes do not display a criticalconcentration above which self-aggregation occurs as found forsurfactants and lipids forming miceller, lamellar or other well definedmeso-phases. Instead, many hydrotropes show a continuous-typeaggregation process where the sizes of aggregates grow as concentrationincreases. However, many hydrotropes alter the phase behavior,stability, and colloidal properties of systems containing substances ofpolar and non-polar character, including mixtures of water, oil,surfactants, and polymers. Hydrotropes are classically used acrossindustries from pharma, personal care, food, to technical applications.Use of hydrotropes in detergent compositions allow for example moreconcentrated formulations of surfactants (as in the process ofcompacting liquid detergents by removing water) without inducingundesired phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt. %, such as from 0 to 5 wt. %,0.5 to 5 wt. %, or from 3 to 5 wt. %, of a hydrotrope. Any hydrotropeknown in the art for use in detergents may be utilized. Non-limitingexamples of hydrotropes include sodium benzenesulfonate, sodiump-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols andpolyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders—The compositions of the present invention may comprise one ormore builders, co-builders, builder systems or a mixture thereof. When abuilder is used, the cleaning composition will typically comprise from 0to 65 wt. %, at least 1 wt. %, from 2 to 60 wt. % or from 5 to 10 wt. %builder. In a dish wash cleaning composition, the level of builder istypically 40 to 65 wt. % or 50 to 65 wt. %. The composition may besubstantially free of builder; substantially free means “no deliberatelyadded” zeolite and/or phosphate. Typical zeolite builders includezeolite A, zeolite P and zeolite MAP. A typical phosphate builder issodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g., a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTM PA),diethylenetriaminepentakis(methylene)pentakis(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N, N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO 2009/102854, U.S. Pat. No. 5,977,053.

In one aspect, the invention relates to compositions comprising a lipasevariant of a parent lipase comprising a substitution at one or more(e.g., several) positions corresponding to positions F7H/K/R,F51A/I/L/V/Y, T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P,P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W,A257F/H/I/L/V/Y, L259F/Y, W260D/E/F/H/I/L/N/Q/S/T/Y of the parentlipase, wherein the variant has lipase activity and has at least 60% butless than 100% sequence identity to the mature polypeptide of the parentlipase or a fragment thereof with lipase activity, the compositioncomprising up to 10 wt. % or 15 wt. % aluminosilicate (anhydrous basis)and/or phosphate builder, the composition having a reserve alkalinity ofgreater than 4 or 7.5. In one aspect the parent lipase is SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO:12. As used herein the term “reserve alkalinity” is a measure of thebuffering capacity of the composition (g/NaOH/100 g composition)determined by titrating a 1% (w/v) solution of composition withhydrochloric acid to pH 7.5, i.e., in order to calculate reservealkalinity. Reserve alkalinity may be calculated as disclosed on page 9in WO 2006/090335. In one aspect the invention relates to compositionscomprising lipase variants further comprising one or more (e.g.,several) substitutions corresponding to any of positions selected from:1, 2, 4, 27, 33, 38, 54, 56, 57, 58, 60, 69, 83, 86, 91, 94, 97, 98, 99,101, 111, 163, 176, 210, 216, 220, 225, 227, 231, 233, 249, 254, 255,256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12. In one aspectthe invention relates to compositions comprising lipase variants furthercomprising one or more (e.g., several) substitutions corresponding toany of positions selected from: E1C, V2K/Y, Q4V, D27R, N33K/Q, G38A,S54T, E56K, D57G, S58A, V60S, L69R, S83T, 186V, G91A/N/Q/T, N94K/R,D96E/G/L/W, L97M, K98E/I, E99K, N101D, D111A, G163K, V176L, E210K/Q/R,S216P, Y220F, G225R, L227G, T231R, N233C/R, Q249R, D254S, P256V, G263Q,L264A, I265T, G266D, T267A, and L269N of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12.

Chelating Agents and Crystal Growth Inhibitors—The compositions hereinmay contain a chelating agent and/or a crystal growth inhibitor.Suitable molecules include copper, iron and/or manganese chelatingagents and mixtures thereof. Suitable molecules include DTPA (Diethylenetriamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid),DTPMP (Diethylene triamine penta(methylene phosphonic acid)),1,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate,ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid(EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEI DA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005to 15 wt. % or from 3.0 to 10 wt. % chelating agent or crystal growthinhibitor.

Bleach Component—The bleach component suitable for incorporation in themethods and compositions of the invention comprise one or a mixture ofmore than one bleach component. Suitable bleach components includebleaching catalysts, photobleaches, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, pre-formed peracids and mixturesthereof. In general, when a bleach component is used, the compositionsof the present invention may comprise from 0 to 30 wt. %, from 0.00001to 90 wt. %, 0.0001 to 50 wt. %, from 0.001 to 25 wt. % or from 1 to 20wt. %. Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular ε-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt. % or from 0.1 to 20 wt. %.

(2) Sources of hydrogen peroxide include, e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt. % or 1 to 30 wt. % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt. % or 0.1 to 20 wt. %.

(3) The term bleach activator is meant herein as a compound which reactswith hydrogen peroxide to form a peracid via perhydrolysis. The peracidthus formed constitutes the activated bleach. Suitable bleach activatorsto be used herein include those belonging to the class of esters,amides, imides or anhydrides. Suitable bleach activators are thosehaving R—(C═O)-L wherein R is an alkyl group, optionally branched,having, when the bleach activator is hydrophobic, from 6 to 14 carbonatoms, or from 8 to 12 carbon atoms and, when the bleach activator ishydrophilic, less than 6 carbon atoms or less than 4 carbon atoms; and Lis leaving group. Examples of suitable leaving groups are benzoic acidand derivatives thereof—especially benzene sulphonate. Suitable bleachactivators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO 98/17767. A family of bleach activators is disclosed in EP624154and particularly preferred in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmentally friendly. Furthermore acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally ATC ismultifunctional, as the citrate released in the perhydrolysis reactionmay function as a builder. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). Suitable bleach activators arealso disclosed in WO 98/17767. While any suitable bleach activator maybe employed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt. %, 0.5 to 40 wt. % or 0.6 to10 wt. % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt. %, or 0.1 to 20 wt. %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g., —N⁺(CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g., —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆₋₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both R¹ and R² are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred aspect the DAP may be asymmetric, such that preferablythe hydrolysis of R1 acyl group is rapid to generate peracid, but thehydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n-C(O)—OO—C(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredaspect, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2): 423-38 (e.g., compound 4, p.433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, preparedas described in U.S. Pat. No. 5,360,569 (e.g., Column 11, Example 1);and N-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (e.g., Column 10, Ex. 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (e.g., Column 31, Ex. II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (e.g., Column 32, Ex. V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO 2005/047264 (e.g., p. 18, Ex.8), and 2[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987): 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4): 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22):2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in the Polish Journal of Chemistry(2003), 77(5): 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Ex. 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34): 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In one aspect, the detergentcomposition comprises a bleach component having a log P_(o/w) no greaterthan 0, no greater than −0.5, no greater than −1.0, no greater than−1.5, no greater than −2.0, no greater than −2.5, no greater than −3.0,or no greater than −3.5. The method for determining log P_(o/w) isdescribed in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety—CR¹¹R¹²—Y-G_(b)-Y_(c)—[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubsituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁹ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one aspect of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt. %, from 0.5 to 40wt. % or from 0.6 to 10 wt. % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO 2009/839406, U.S. Pat. No.6,218,351 and WO 00/12667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described,e.g., in U.S. Pat. Nos. 5,597,936; 5,595,967. Such cobalt catalysts arereadily prepared by known procedures, such as taught, e.g., in U.S. Pat.Nos. 5,597,936 and 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include, e.g., manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught, e.g., in U.S. Pat. No. 6,225,464 and WO 00/32601.

(7) Photobleaches—suitable photobleaches include, e.g., sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Exemplary bleaching systems are also described, e.g., in WO 2007/087258,WO 2007/087244, WO 2007/087259 and WO 2007/087242.

Fabric Hueing Agents—The composition may comprise a fabric hueing agent.Suitable fabric hueing agents include dyes, dye-clay conjugates, andpigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Color Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof.

In one aspect, suitable small molecule dyes include small molecule dyesselected from the group consisting of Color Index (Society of Dyers andColorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In one aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In one aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In one aspect, suitable polymeric dyes include polymeric dyes selectedfrom the group consisting of fabric-substantive colorants sold under thename of Liquitint® (Milliken), dye-polymer conjugates formed from atleast one reactive dye and a polymer selected from the group consistingof polymers comprising a moiety selected from the group consisting of ahydroxyl moiety, a primary amine moiety, a secondary amine moiety, athiol moiety and mixtures thereof. In still one aspect, suitablepolymeric dyes include polymeric dyes selected from the group consistingof Liquitint® Violet CT, carboxymethyl cellulose (CMC) conjugated with areactive blue, reactive violet or reactive red dye such as CMCconjugated with C.I. Reactive Blue 19, sold by Megazyme, Wicklow,Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC,alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophenepolymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO2008/087497. These whitening agents may be characterized by thefollowing structure (I):

wherein R₁ and R₂ can independently be selected from:

-   -   a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]        wherein R′ is selected from the group consisting of H, CH₃,        CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected        from the group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures        thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;    -   b) R₁=alkyl, aryl or aryl alkyl and        R₂═[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]        wherein R′ is selected from the group consisting of H, CH₃,        CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected        from the group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures        thereof; wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;    -   c) R₁═[CH₂CH₂(OR₃)CH₂OR₄] and R₂═[CH₂CH₂(O R₃)CH₂O R₄]        wherein R₃ is selected from the group consisting of H,        (CH₂CH₂O)_(z)H, and mixtures thereof; and        wherein z=0 to 10;        wherein R₄ is selected from the group consisting of        (C₁-C₁₆)alkyl, aryl groups, and mixtures thereof; and    -   d) wherein R1 and R2 can independently be selected from the        amino addition product of styrene oxide, glycidyl methyl ether,        isobutyl glycidyl ether, isopropylglycidyl ether, t-butyl        glycidyl ether, 2-ethylhexylgycidyl ether, and glycidylhexadecyl        ether, followed by the addition of from 1 to 10 alkylene oxide        units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 1 R1 R2 R′ R″ X y R′ R″ X y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1

Further whitening agents of use include those described in US2008/0034511 (Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In one aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of onecationic/basic dye selected from the group consisting of C.I. BasicYellow 1 through 108, C.I. Basic Orange 1 through 69, C.I. Basic Red 1through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue 1 through164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through 23, CIBasic Black 1 through 11, and a clay selected from the group consistingof Montmorillonite clay, Hectorite clay, Saponite clay and mixturesthereof. In still one aspect, suitable dye clay conjugates include dyeclay conjugates selected from the group consisting of: MontmorilloniteBasic Blue B7 C.I. 42595 conjugate, Montmorillonite Basic Blue B9 C.I.52015 conjugate, Montmorillonite Basic Violet V3 C.I. 42555 conjugate,Montmorillonite Basic Green G1 C.I. 42040 conjugate, MontmorilloniteBasic Red R1 C.I. 45160 conjugate, Montmorillonite C.I. Basic Black 2conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate, Hectorite BasicBlue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3 C.I. 42555conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate, HectoriteBasic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black 2conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In one aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (C.I. Pigment Blue 29), UltramarineViolet (C.I. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt. %, or 0.0001to 0.25 wt. %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/I.

Encapsulates—The composition may comprise an encapsulate. In one aspect,an encapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or 90% of said encapsulates may have afracture strength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to3.5 MPa, or from 0.7 to 3 MPa; and a benefit agent leakage of from 0 to30%, from 0 to 20%, or from 0 to 5%.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle size from 1 to 80 microns, from 5 to 60 microns, from 10 to 50microns, or from 15 to 40 microns.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle wall thickness from 30 to 250 nm, from 80 to 180 nm, or from100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates, e.g., in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching of US2008/0305982; and/or US 2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—In one aspect the composition comprises a perfume thatcomprises one or more perfume raw materials selected from the groupconsisting of 1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid,diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol,monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid, 2-hydroxy-,3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal,4-ethyl-alpha,alpha-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,gamma-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificaspects of the amphiphilic alkoxylated grease cleaning polymers of thepresent invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO 91/08281 and WO 90/01815. Chemically,these materials comprise polyacrylates having one ethoxy side-chain perevery 7-8 acrylate units. The side-chains are of the formula—(CH₂CH₂O)_(m) (CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. Theside-chains are ester-linked to the polyacrylate “backbone” to provide a“comb” polymer type structure. The molecular weight can vary, but istypically in the range of 2000 to 50,000. Such alkoxylatedpolycarboxylates can comprise from 0.05 to 10 wt. % of the compositionsherein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate polymer—The composition of the present invention may alsoinclude one or more carboxylate polymers such as a maleate/acrylaterandom copolymer or polyacrylate homopolymer. In one aspect, thecarboxylate polymer is a polyacrylate homopolymer having a molecularweight of from 4,000 to 9,000 Da, or from 6,000 to 9,000 Da.

Soil release polymer—The composition of the present invention may alsoinclude one or more soil release polymers having a structure as definedby one of the following structures (I), (II) or (III):—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)  (I)—[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO—]_(e)  (II)—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)  (III)wherein:

-   -   a, b and c are from 1 to 200;    -   d, e and f are from 1 to 50;    -   Ar is a 1,4-substituted phenylene;    -   sAr is 1,3-substituted phenylene substituted in position 5 with        SO₃Me;    -   Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, or        tetraalkylammonium wherein the alkyl    -   groups are C₁-C₁₈ alkyl or C₂-C₁₀ hydroxyalkyl, or mixtures        thereof;    -   R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or        C₁-C₁₈ n- or iso-alkyl; and    -   R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched        C₂-C₃₀ alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms,        or a C₈-C₃₀ aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic polymer—The composition of the present invention may alsoinclude one or more cellulosic polymers including those selected fromalkyl cellulose, alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose,alkyl carboxyalkyl cellulose. In one aspect, the cellulosic polymers areselected from the group comprising carboxymethyl cellulose, methylcellulose, methyl hydroxyethyl cellulose, methyl carboxymethylcellulose, and mixures thereof. In one aspect, the carboxymethylcellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 anda molecular weight from 100,000 to 300,000 Da.

Enzymes—The composition may comprise one or more enzymes which providecleaning performance and/or fabric care benefits. Examples of suitableenzymes include, but are not limited to, hemicellulases, peroxidases,proteases, cellulases, xylanases, lipases, phospholipases, esterases,cutinases, pectinases, mannanases, pectate lyases, keratinases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, malanases, β-glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases,amylases, or mixtures thereof. A typical combination is an enzymecocktail that may comprise, e.g., a protease and lipase in conjunctionwith amylase. When present in a composition, the aforementionedadditional enzymes may be present at levels from 0.00001 to 2 wt. %,from 0.0001 to 1 wt. % or from 0.001 to 0.5 wt. % enzyme protein byweight of the composition.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

In one aspect preferred enzymes would include a cellulase. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 495257, EP 531372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0531315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

In one aspect preferred enzymes would include a protease. Suitableproteases include those of bacterial, fungal, plant, viral or animalorigin, e.g., vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from, e.g., family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezen et al.,1997, Protein Science 6: 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e., the Subtilisin family, theThermitase family, the Proteinase K family, the Lantibiotic peptidasefamily, the Kexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in U.S. Pat. No. 7,262,042 andWO 2009/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO 89/06279 and proteasePD138 described in (WO 93/18140). Other useful proteases may be thosedescribed in WO 92/175177, WO 01/16285, WO 02/026024 and WO 02/016547.Examples of trypsin-like proteases are trypsin (e.g., of porcine orbovine origin) and the Fusarium protease described in WO 89/06270, WO94/25583 and WO 2005/040372, and the chymotrypsin proteases derived fromCellumonas described in WO 2005/052161 and WO 2005/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO 95/23221, and variantsthereof which are described in WO 92/21760, WO 95/23221, EP 1921147 andEP 1921148,

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO 2007/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO 92/19729,WO 96/034946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO03/006602, WO 2004/03186, WO 2004/041979, WO 2007/006305, WO2011/036263, WO 2011/036264, especially the variants with substitutionsin one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68,76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123,128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222,224, 232, 235, 236, 245, 248, 252 and 274 using the BPN′ numbering. Morepreferred the subtilase variants may comprise the mutations: S3T, V4I,S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD,S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L,P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D,N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN'numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Blaze®; Duralase™, Durazym™, Relase®,Savinase®, Primase®, Polarzyme®, Kannase®, Liquanase®, Ovozyme®,Coronase®, Neutrase®, Everlase® and Esperase® all could be sold asUltra® or Evity® (Novozymes A/S), those sold under the tradenameMaxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™,Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®,Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

In one aspect, preferred enzymes would include an amylase. Suitableamylases may be an alpha-amylase or a glucoamylase and may be ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB 1296839.

Suitable amylases include amylases having SEQ ID NO: 3 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/19467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions:

-   -   M197T;    -   H156Y+A181T+N190F+A209V+Q264S; or    -   G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 SEQ ID NO:7; or SEQ ID NO: 9 of WO 96/23873 orvariants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO:7, or SEQ ID NO: 9. Preferred variants ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO:7; or SEQ ID NO: 9are those having a substitution, a deletion or an insertion in one ormore of the following positions: 140, 181, 182, 183, 184, 195, 206, 212,243, 260, 269, 304 and 476. More preferred variants are those having adeletion in positions 181 and 182 or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO:7; orSEQ ID NO: 9 are those having a deletion in positions 183 and 184 and asubstitution in one or more of positions 140, 195, 206, 243, 260, 304and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 of WO2008/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 2008/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO2009/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:

-   -   N128C+K178L+T182G+Y305R+G475K;    -   N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;    -   S125A+N128C+K178L+T182G+Y305R+G475K; or    -   S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K        wherein the variants are C-terminally truncated and optionally        further comprises a substitution at position 243 and/or a        deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO 01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO 2013/001078 and WO 2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, TermamylUltra™, Fungamyl™, Ban™, Stainzyme™, Stainzyme Plus™, Amplify®,Supramyl™, Natalase™ Liquozyme X, BAN™, Resillience and Everest (fromNovozymes A/S), KEMZYM® AT 9000 Biozym Biotech Trading GmbH Wehlistrasse27b A-1200 Wien Austria, and Rapidase™ Purastar™/Effectenz™, Powerase,Preferenz S100, Preferenx S110, ENZYSIZE®, OPTISIZE HT PLUS®, andPURASTAR OXAM® (Danisco/DuPont) and KAM® (Kao).

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g., from T.lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamedto Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyceslipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO2010/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No.5,389,536), lipase from Thermobifida fusca (WO 2011/084412, WO2013/033318), Geobacillus stearothermophilus lipase (WO 2011/084417),lipase from Bacillus subtilis (WO 2011/084599), and lipase fromStreptomyces griseus (WO 2011/150157) and S. pristinaespiralis (WO2012/137147).

Other examples are lipase variants such as those described in EP 407225,WO 92/05249, WO 94/01541, WO 94/25578, WO 95/14783, WO 95/30744, WO95/35381, WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.

Preferred commercial lipase products include Lipolase™, Lipex™, LipexEvity™, Lipolex™, and Lipoclean™ (Novozymes A/S), Lumafast (originallyfrom Genencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g., acyltransferases with homologyto Candida antarctica lipase A (WO 2010/111143), acyltransferase fromMycobacterium smegmatis (WO 2005/56782), perhydrolases from the CE 7family (WO 2009/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4),including a bacterial polypeptide endogenous to a member of the genusBacillus which has a sequence of at least 90%, 94%, 97% or 99% identityto the amino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403 andmixtures thereof. Suitable endoglucanases are sold under the tradenamesCelluclean® and Whitezyme® (Novozymes).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco/DuPont).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as granulate, liquid, slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

Dye Transfer Inhibiting Agents—The compositions of the present inventionmay also include one or more dye transfer inhibiting agents. Suitablepolymeric dye transfer inhibiting agents include, but are not limitedto, polyvinylpyrrolidone polymers, polyamine N-oxide polymers,copolymers of N-vinylpyrrolidone and N-vinylimidazole,polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Whenpresent in a composition, the dye transfer inhibiting agents may bepresent at levels from 0.0001 to 10 wt. %, from 0.01 to 5 wt. % or from0.1 to 3 wt. %.

Brighteners—The compositions of the present invention can also containadditional components that may tint articles being cleaned, such asfluorescent brighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt. %, at least 75 wt. %, at least 90wt. %, at least 99 wt. %, or even substantially all, of the C.I.fluorescent brightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making 0.1 fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. Nos. 4,790,856 and 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt. %, from 0.05 wt. %, from 0.1 wt. % or from 0.2 wt. % to upper levelsof 0.5 wt. % or 0.75 wt. %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts—The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 to less than 10 wt. % silicate salt,to 9 wt. %, or to 8 wt. %, or to 7 wt. %, or to 6 wt. %, or to 5 wt. %,or to 4 wt. %, or to 3 wt. %, or even to 2 wt. %, and from above 0 wt.%, or from 0.5 wt. %, or from 1 wt. % silicate salt. A suitable silicatesalt is sodium silicate.

Dispersants—The compositions of the present invention can also containdispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—Enzymes for use in compositions can be stabilized byvarious techniques. The enzymes employed herein can be stabilized by thepresence of water-soluble sources of calcium and/or magnesium ions.Examples of conventional stabilizing agents are, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, a peptidealdehyde, lactic acid, boric acid, or a boric acid derivative, e.g., anaromatic borate ester, or a phenyl boronic acid derivative such as4-formylphenyl boronic acid, and the composition may be formulated asdescribed in, for example, WO 92/19709 and WO 92/19708. In case ofaqueous compositions comprising protease, a reversible proteaseinhibitor, such as a boron compound including borate, 4-formylphenylboronic acid, phenylboronic acid and derivatives thereof, orcompounds such as calcium formate, sodium formate and 1,2-propane diolcan be added to further improve stability. The peptide aldehyde may beof the formula B₂—B₁—B₀—R wherein: R is hydrogen, CH₃, CX₃, CHX₂, orCH₂X, wherein X is a halogen atom; B₀ is a phenylalanine residue with anOH substituent at the p-position and/or at the m-position; B₁ is asingle amino acid residue; and B₂ consists of one or more amino acidresidues, optionally comprising an N-terminal protection group.Preferred peptide aldehydes include but are not limited to thosedescribed in WO 2009/118375, WO 2011/036153, and WO 2013/004636 such ase.g Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H,Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H or Ac-WLVY-H, whereZ is benzyloxycarbonyl and Ac is acetyl. Examples of other suitablepeptide aldehydes include a-MAPI, P-MAPI, F-urea-RVY-H, F-urea-GGY-H,F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H,F-urea-GA-Nle-H, Y-urea-RVY-H, Y-urea-GAY-H, F-CS-RVF-H, F-CS-RVY-H,F-CS-GAY-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B,and Chymostatin C. Further examples of peptide aldehydes are disclosedin WO 2010/055052, WO 2009/118375, WO 94/04651, WO 98/13459, WO98/13461, WO 98/13462, and WO 2007/145963.

Solvents—Suitable solvents include water and other solvents such aslipophilic fluids. Examples of suitable lipophilic fluids includesiloxanes, other silicones, hydrocarbons, glycol ethers, glycerinederivatives such as glycerine ethers, perfluorinated amines,perfluorinated and hydrofluoroether solvents, low-volatilitynonfluorinated organic solvents, diol solvents, otherenvironmentally-friendly solvents and mixtures thereof.

Structurant/Thickeners—Structured liquids can either be internallystructured, whereby the structure is formed by primary ingredients(e.g., surfactant material) and/or externally structured by providing athree dimensional matrix structure using secondary ingredients (e.g.,polymers, clay and/or silicate material). The composition may comprise astructurant, from 0.01 to 5 wt. %, or from 0.1 to 2.0 wt. %. Thestructurant is typically selected from the group consisting ofdiglycerides and triglycerides, ethylene glycol distearate,microcrystalline cellulose, cellulose-based materials, microfibercellulose, hydrophobically modified alkali-swellable emulsions such asPolygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, andmixtures thereof. A suitable structurant includes hydrogenated castoroil, and non-ethoxylated derivatives thereof. A suitable structurant isdisclosed in U.S. Pat. No. 6,855,680. Such structurants have athread-like structuring system having a range of aspect ratios. Othersuitable structurants and the processes for making them are described inWO 2010/034736.

Conditioning Agents—The composition of the present invention may includea high melting point fatty compound. The high melting point fattycompound useful herein has a melting point of 25° C. or higher, and isselected from the group consisting of fatty alcohols, fatty acids, fattyalcohol derivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1 to 40 wt. %, from 1 to 30 wt. %, from 1.5 to 16 wt.%, from 1.5 to 8 wt. % in view of providing improved conditioningbenefits such as slippery feel during the application to wet hair,softness and moisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3 wt. %, from 0.075 to 2.0 wt. %, or from0.1 to 1.0 wt. %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH 3 to pH 9, or between pH 4 and pH 8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. Nos. 3,962,418; 3,958,581;and US 2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt. %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. Nos. 5,104,646;5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837; 6,607,717;6,482,969; 5,807,956; 5,981,681; 6,207,782; 7,465,439; 7,041,767;7,217,777; US 2007/0286837; US 2005/0048549; US 2007/0041929; GB 849433;DE 10036533, which are all incorporated herein by reference; Chemistryand Technology of Silicones, New York: Academic Press (1968); GeneralElectric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54 and SE76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

The compositions of the present invention may also comprise from 0.05 to3 wt. % of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. Nos. 5,674,478 and 5,750,122 or in U.S. Pat. Nos.4,529,586; 4,507,280; 4,663,158; 4,197,865; 4,217,914; 4,381,919; and4,422,853.

Hygiene and malodour—The compositions of the present invention may alsocomprise one or more of zinc ricinoleate, thymol, quaternary ammoniumsalts such as Bardac®, polyethylenimines (such as Lupasol® from BASF)and zinc complexes thereof, silver and silver compounds, especiallythose designed to slowly release Ag⁺ or nano-silver dispersions.

Probiotics—The compositions may comprise probiotics such as thosedescribed in WO 2009/043709.

Suds Boosters—If high sudsing is desired, suds boosters such as theC₁₀-C₁₆ alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporatedinto the compositions, typically at 1 to 10 wt. % levels. The C₁₀-C₁₄monoethanol and diethanol amides illustrate a typical class of such sudsboosters. Use of such suds boosters with high sudsing adjunctsurfactants such as the amine oxides, betaines and sultaines noted aboveis also advantageous. If desired, water-soluble magnesium and/or calciumsalts such as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added atlevels of, typically, 0.1 to 2 wt. %, to provide additional suds and toenhance grease removal performance.

Suds Suppressors—Compounds for reducing or suppressing the formation ofsuds can be incorporated into the compositions of the present invention.Suds suppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. Nos. 4,489,455and 4,489,574, and in front-loading-style washing machines. A widevariety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See, e.g., KirkOthmer Encyclopedia of Chemical Technology, Third Edition, Volume 7, p.430-447 (John Wiley & Sons, Inc., 1979). Examples of suds supressorsinclude monocarboxylic fatty acid and soluble salts therein, highmolecular weight hydrocarbons such as paraffin, fatty acid esters (e.g.,fatty acid triglycerides), fatty acid esters of monovalent alcohols,aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated amino triazines,waxy hydrocarbons preferably having a melting point below about 100° C.,silicone suds suppressors, and secondary alcohols. Suds supressors aredescribed in U.S. Pat. Nos. 2,954,347; 4,265,779; 4,265,779; 3,455,839;3,933,672; 4,652,392; 4,978,471; 4,983,316; 5,288,431; 4,639,489;4,749,740; 4,798,679; 4,075,118; EP 89307851.9; EP 150872; and DOS2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt. % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt. %. Preferably, from 0.5 to 3 wt. % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt. %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2 wt. %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt. %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3 wt. %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain aspects the compositions have cleaning activity frompH 4 to pH 11.5. In other aspects, the compositions are active from pH 6to pH 11, from pH 7 to pH 11, from pH 8 to pH 11, from pH 9 to pH 11, orfrom pH 10 to pH 11.5.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainaspects, the optimum temperature range for the compositions is from 10°C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20° C.to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C. to45° C., or from 40° C. to 50° C.

Form of the Composition

The compositions described herein are advantageously employed forexample, in laundry applications, hard surface cleaning, dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. The compositions of the invention are in particular solidor liquid cleaning and/or treatment compositions. In one aspect theinvention relates to a composition, wherein the form of the compositionis selected from the group consisting of a regular, compact orconcentrated liquid; a gel; a paste; a soap bar; a regular or acompacted powder; a granulated solid; a homogenous or a multilayertablet with two or more layers (same or different phases); a pouchhaving one or more compartments; a single or a multi-compartment unitdose form; or any combination thereof.

The form of the composition may separate the components physically fromeach other in compartments such as, e.g., water dissolvable pouches orin different layers of tablets. Thereby negative storage interactionbetween components can be avoided. Different dissolution profiles ofeach of the compartments can also give rise to delayed dissolution ofselected components in the wash solution.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g., without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids (US 2009/0011970).

Lipase Particles

The lipase variants of the present invention may be comprised inwater-soluble film as lipase particles. The lipase particles may containone or more additional enzymes, as described below.

Lipase particles are any form of lipase variant in a solid particulateform. That can be as lipase crystals, lipase precipitate, spray orfreeze-dried lipase or any form of granulated lipase, either as a powderor a suspension in liquid. Typically the particle size, measured asequivalent spherical diameter (volume based average particle size), ofthe lipase particles is below 2 mm, preferably below 1 mm, below 0.5 mm,below 0.25 mm, or below 0.1 mm; and above 0.05 um, preferably above 0.1um, above 0.5 um, above 1 um, above 5 um or above 10 um. In a preferredembodiment, the particle size of the lipase particles is from 0.5 um to100 um.

The lipase particles contain at least 1% w/w lipase protein, preferablyat least 5% w/w lipase protein, at least 10% w/w lipase protein, atleast 20% w/w lipase protein, at least 30% w/w lipase protein, at least40% w/w lipase protein, at least 50% w/w lipase protein, at least 60%w/w lipase protein, at least 70% w/w lipase protein, at least 80% w/wlipase protein, or at least 90% w/w lipase protein.

In a preferred embodiment, the lipase particles are lipase crystals, orthe lipase protein is on a crystalline form. Enzyme crystallization maybe carried out in a number of ways, as known in the art (e.g., asdescribed in WO 91/09943 or WO 94/22903).

The lipase may be formulated in the lipase particle as known in the artfor solid enzyme formulations, such as formulations for reducing dust,improving stability and/or modifying relase rate of the enzyme. Thelipase particle may also be formulated in a matrix or coated with agentssuppressing dissolution of the enzyme particle in the PVOH/film solutionused for preparing the water-soluble film.

The lipase molecules on the surface of the lipase particles may also becross-linked, like CLECs (Cross-Linked Enzyme Crystals) or CLEA(Cross-Linked Enzyme Aggregate).

Water-Soluble Film

Water-soluble films, optional ingredients for use therein, and methodsof making the same are well known in the art. In one class ofembodiments, the water-soluble film includes PVOH. PVOH is a syntheticresin generally prepared by the alcoholysis, usually termed hydrolysisor saponification, of polyvinyl acetate. Fully hydrolyzed PVOH, whereinvirtually all the acetate groups have been converted to alcohol groups,is a strongly hydrogen-bonded, highly crystalline polymer whichdissolves only in hot water—greater than about 140° F. (60° C.). If asufficient number of acetate groups are allowed to remain after thehydrolysis of polyvinyl acetate, the PVOH polymer then being known aspartially hydrolyzed, it is more weakly hydrogen-bonded and lesscrystalline and is soluble in cold water—less than about 50° F. (10°C.). An intermediate cold/hot water-soluble film can include, forexample, intermediate partially-hydrolyzed PVOH (e.g., with degrees ofhydrolysis of about 94% to about 98%), and is readily soluble only inwarm water—e.g., rapid dissolution at temperatures of about 40° C. andgreater. Both fully and partially hydrolyzed PVOH types are commonlyreferred to as PVOH homopolymers although the partially hydrolyzed typeis technically a vinyl alcohol-vinyl acetate copolymer.

The degree of hydrolysis of the PVOH included in the water-soluble filmsmay be about 75% to about 99%. As the degree of hydrolysis is reduced, afilm made from the resin will have reduced mechanical strength butfaster solubility at temperatures below about 20° C. As the degree ofhydrolysis increases, a film made from the resin will tend to bemechanically stronger and the thermoformability will tend to decrease.The degree of hydrolysis of the PVOH can be chosen such that thewater-solubility of the resin is temperature dependent, and thus thesolubility of a film made from the resin, compatibilizing agent, andadditional ingredients is also influenced. In one class of embodimentsthe film is cold water-soluble. A cold water-soluble film, soluble inwater at a temperature of less than 10° C., can include PVOH with adegree of hydrolysis in a range of about 75% to about 90%, or in a rangeof about 80% to about 90%, or in a range of about 85% to about 90%. Inanother class of embodiments the film is hot water-soluble. A hotwater-soluble film, soluble in water at a temperature of at least about60° C., can include PVOH with a degree of hydrolysis of at least about98%.

Other film-forming resins for use in addition to or in an alternative toPVOH can include, but are not limited to, modified polyvinyl alcohols,polyacrylates, water-soluble acrylate copolymers, polyacrylates,polyacryamides, polyvinyl pyrrolidone, pullulan, water-soluble naturalpolymers including, but not limited to, guar gum, xanthan gum,carrageenan, and starch, water-soluble polymer derivatives including,but not limited to, ethoxylated starch and hydroxypropylated starch,poly(sodium acrylamido-2-methylpropane sulfonate),polymonomethylmaleate, copolymers thereof, and combinations of any ofthe foregoing. In one class of embodiments, the film-forming resin is aterpolymer consisting of vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate. Unexpectedly, water-soluble filmsbased on a vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate terpolymer have demonstrated a highpercent recovery of enzyme.

The water-soluble resin can be included in the water-soluble film in anysuitable amount, for example an amount in a range of about 35 wt. % toabout 90 wt. %. The preferred weight ratio of the amount of thewater-soluble resin as compared to the combined amount of all enzymes,enzyme stabilizers, and secondary additives can be any suitable ratio,for example a ratio in a range of about 0.5 to about 5, or about 1 to 3,or about 1 to 2.

Water-soluble resins for use in the films described herein (including,but not limited to PVOH resins) can be characterized by any suitableviscosity for the desired film properties, optionally a viscosity in arange of about 5.0 to about 30.0 cP, or about 10.0 cP to about 25 cP.The viscosity of a PVOH resin is determined by measuring a freshly madesolution using a Brookfield LV type viscometer with UL adapter asdescribed in British Standard EN ISO 15023-2:2006 Annex E BrookfieldTest method. It is international practice to state the viscosity of 4%aqueous polyvinyl alcohol solutions at 20° C. All PVOH viscositiesspecified herein in cP should be understood to refer to the viscosity of4% aqueous polyvinyl alcohol solution at 20° C., unless specifiedotherwise.

It is well known in the art that the viscosity of a PVOH resin iscorrelated with the weight average molecular weight of the same PVOHresin, and often the viscosity is used as a proxy for the weight averagemolecular weight. Thus, the weight average molecular weight of thewater-soluble resin optionally can be in a range of about 35,000 toabout 190,000, or about 80,000 to about 160,000. The molecular weight ofthe resin need only be sufficient to enable it to be molded by suitabletechniques to form a thin plastic film.

The water-soluble films may include other optional additive ingredientsincluding, but not limited to, plasticizers, surfactants, defoamers,film formers, antiblocking agents, internal release agents,anti-yellowing agents and other functional ingredients, for example inamounts suitable for their intended purpose.

Water is recognized as a very efficient plasticizer for PVOH and otherpolymers; however, the volatility of water makes its utility limitedsince polymer films need to have at least some resistance (robustness)to a variety of ambient conditions including low and high relativehumidity. Glycerin is much less volatile than water and has been wellestablished as an effective plasticizer for PVOH and other polymers.Glycerin or other such liquid plasticizers by themselves can causesurface “sweating” and greasiness if the level used in the filmformulation is too high. This can lead to problems in a film such asunacceptable feel to the hand of the consumer and even blocking of thefilm on the roll or in stacks of sheets if the sweating is not mitigatedin some manner, such as powdering of the surface. This could becharacterized as over plasticization. However, if too little plasticizeris added to the film the film may lack sufficient ductility andflexibility for many end uses, for example to be converted into a finaluse format such as pouches.

Plasticizers for use in water-soluble films of the present disclosureinclude, but are not limited to, sorbitol, glycerol, diglycerol,propylene glycol, ethylene glycol, diethyleneglycol, triethylene glycol,tetraethyleneglycol, polyethylene glycols up to MW 400, 2 methyl 1,3propane diol, lactic acid, monoacetin, triacetin, triethyl citrate,1,3-butanediol, trimethylolpropane (TMP), polyether triol, andcombinations thereof. Polyols, as described above, are generally usefulas plasticizers. As less plasticizer is used, the film can become morebrittle, whereas as more plasticizer is used the film can lose tensilestrength. Plasticizers can be included in the water-soluble films in anamount in a range of about 25 phr to about 50 phr, or from about 30 phrto about 45 phr, or from about 32 phr to about 42 phr, for example.

Surfactants for use in water-soluble films are well known in the art.Optionally, surfactants are included to aid in the dispersion of theresin solution upon casting. Suitable surfactants for water-solublefilms of the present disclosure include, but are not limited to, dialkylsulfosuccinates, lactylated fatty acid esters of glycerol and propyleneglycol, lactylic esters of fatty acids, sodium alkyl sulfates,polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkylpolyethylene glycol ethers, lecithin, acetylated fatty acid esters ofglycerol and propylene glycol, sodium lauryl sulfate, acetylated estersof fatty acids, myristyl dimethylamine oxide, trimethyl tallow alkylammonium chloride, quaternary ammonium compounds, salts thereof andcombinations of any of the forgoing. Thus, surfactants can be includedin the water-soluble films in an amount of less than about 2 phr, forexample less than about 1 phr, or less than about 0.5 phr, for example.

One type of secondary component contemplated for use is a defoamer.Defoamers can aid in coalescing of foam bubbles. Suitable defoamers foruse in water-soluble films according to the present disclosure include,but are not limited to, hydrophobic silicas, for example silicon dioxideor fumed silica in fine particle sizes, including Foam Blast® defoamersavailable from Emerald Performance Materials, including Foam Blast® 327,Foam Blast® UVD, Foam Blast® 163, Foam Blast® 269, Foam Blast® 338, FoamBlast® 290, Foam Blast® 332, Foam Blast® 349, Foam Blast® 550 and FoamBlast® 339, which are proprietary, non-mineral oil defoamers. Inembodiments, defoamers can be used in an amount of 0.5 phr, or less, forexample, 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr.Preferably, significant amounts of silicon dioxide will be avoided, inorder to avoid stress whitening.

Processes for making water-soluble articles, including films, includecasting, blow-molding, extrusion and blown extrusion, as known in theart. One contemplated class of embodiments is characterized by thewater-soluble film described herein being formed by casting, forexample, by admixing the ingredients described herein with water tocreate an aqueous mixture, for example a solution with optionallydispersed solids, applying the mixture to a surface, and drying offwater to create a film. Similarly, other compositions can be formed bydrying the mixture while it is confined in a desired shape.

In one contemplated class of embodiments, the water-soluble film isformed by casting a water-soluble mixture wherein the water-solublemixture is prepared according to the steps of:

-   -   (a) providing a mixture of water-soluble resin, water, and any        optional additives excluding plasticizers;    -   (b) boiling the mixture for 30 minutes;    -   (c) degassing the mixture in an oven at a temperature of at        least 40° C.; optionally in a range of 40° C. to 70° C., e.g.,        about 65° C.;    -   (d) adding one or more enzymes, plasticizer, and additional        water to the mixture at a temperature of 65° C. or less;    -   (e) stirring the mixture without vortex until the mixture        appears substantially uniform in color and consistency;        optionally for a time period in a range of 30 minutes to 90        minutes, optionally at least 1 hour; and    -   (f) casting the mixture promptly after the time period of        stirring (e.g., within 4 hours, or 2 hours, or 1 hour).

If the enzyme is added to the mixture too early, e.g., with thesecondary additives or resin, the activity of the enzyme may decrease.Without intending to be bound by any particular theory, it is believedthat boiling of the mixture with the enzyme leads to the enzymedenaturing and storing in solution for extended periods of time alsoleads to a reduction in enzyme activity.

In one class of embodiments, high enzyme activity is maintained in thewater-soluble films according to the present disclosure by drying thefilms quickly under moderate to mild conditions. As used herein, dryingquickly refers to a drying time of less than 24 hours, optionally lessthan 12 hours, optionally less than 8 hours, optionally less than 2hours, optionally less than 1 hour, optionally less than 45 minutes,optionally less than 30 minutes, optionally less than 20 minutes,optionally less than 10 minutes, for example in a range of about 6minutes to about 10 minutes, or 8 minutes. As used herein, moderate tomild conditions refer to drying temperatures of less than 170° F. (77°C.), optionally in a range of about 150° F. to about 170° F. (about 66°C. to about 77° C.), e.g., 165° F. (74° C.). As the drying temperatureincreases, the enzymes tend to denature faster, whereas as the dryingtemperature decreases, the drying time increases, thus exposing theenzymes to solution for an extended period of time.

The film is useful for creating a packet to contain a composition, forexample laundry or dishwashing compositions, thereby forming a pouch.The film described herein can also be used to make a packet with two ormore compartments made of the same film or in combination with films ofother polymeric materials. Additional films can, for example, beobtained by casting, blow-molding, extrusion or blown extrusion of thesame or a different polymeric material, as known in the art. In one typeof embodiment, the polymers, copolymers or derivatives thereof suitablefor use as the additional film are selected from polyvinyl alcohols,polyvinyl pyrrolidone, polyalkylene oxides, polyacrylic acid, cellulose,cellulose ethers, cellulose esters, cellulose amides, polyvinylacetates, polycarboxylic acids and salts, polyaminoacids or peptides,polyamides, polyacrylamide, copolymers of maleic/acrylic acids,polysaccharides including starch and gelatin, natural gums such asxanthan, and carrageenans. For example, polymers can be selected frompolyacrylates and water-soluble acrylate copolymers, methylcellulose,carboxymethylcellulose sodium, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and combinations thereof, or selected from polyvinylalcohols, polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC), and combinations thereof.

The pouches and/or packets may comprise at least one sealed compartment.Thus the pouches may comprise a single compartment or multiplecompartments. The pouches may have regions with and without enzymes. Inembodiments including multiple compartments, each compartment maycontain identical and/or different compositions. In turn, thecompositions may take any suitable form including, but not limited toliquid, solid and combinations thereof (e.g., a solid suspended in aliquid). In some embodiments, the pouches comprises a first, second andthird compartment, each of which respectively contains a differentfirst, second and third composition. In some embodiments, thecompositions may be visually distinct as described in EP 2258820.

The compartments of multi-compartment pouches and/or packets may be ofthe same or different size(s) and/or volume(s). The compartments of thepresent multi-compartment pouches can be separate or conjoined in anysuitable manner. In some embodiments, the second and/or third and/orsubsequent compartments are superimposed on the first compartment. Inone aspect, the third compartment may be superimposed on the secondcompartment, which is in turn superimposed on the first compartment in asandwich configuration. Alternatively the second and third compartmentsmay be superimposed on the first compartment. However it is also equallyenvisaged that the first, second and optionally third and subsequentcompartments may be attached to one another in a side by siderelationship. The compartments may be packed in a string, eachcompartment being individually separable by a perforation line. Henceeach compartment may be individually torn-off from the remainder of thestring by the end-user.

In some embodiments, multi-compartment pouches and/or packets includethree compartments consisting of a large first compartment and twosmaller compartments. The second and third smaller compartments aresuperimposed on the first larger compartment. The size and geometry ofthe compartments are chosen such that this arrangement is achievable.The geometry of the compartments may be the same or different. In someembodiments the second and optionally third compartment each has adifferent geometry and shape as compared to the first compartment. Inthese embodiments, the second and optionally third compartments arearranged in a design on the first compartment. The design may bedecorative, educative, or illustrative, for example to illustrate aconcept or instruction, and/or used to indicate origin of the product.In some embodiments, the first compartment is the largest compartmenthaving two large faces sealed around the perimeter, and the secondcompartment is smaller covering less than about 75%, or less than about50% of the surface area of one face of the first compartment. Inembodiments in which there is a third compartment, the aforementionedstructure may be the same but the second and third compartments coverless than about 60%, or less than about 50%, or less than about 45% ofthe surface area of one face of the first compartment.

The pouches and/or packets may comprise one or more different films. Forexample, in single compartment embodiments, the packet may be made fromone wall that is folded onto itself and sealed at the edges, oralternatively, two walls that are sealed together at the edges. Inmultiple compartment embodiments, the packet may be made from one ormore films such that any given packet compartment may comprise wallsmade from a single film or multiple films having differing compositions.In one aspect, a multi-compartment pouch comprises at least three walls:an outer upper wall; an outer lower wall; and a partitioning wall. Theouter upper wall and the outer lower wall are generally opposing andform the exterior of the pouch. The partitioning wall is interior to thepouch and is secured to the generally opposing outer walls along a sealline. The partitioning wall separates the interior of themulti-compartment pouch into at least a first compartment and a secondcompartment. In one class of embodiments, the partitioning wall may bethe only enzyme containing film thereby minimizing the exposure of theconsumer to the enzymes.

Pouches and packets may be made using any suitable equipment and method.For example, single compartment pouches may be made using vertical formfilling, horizontal form filling, or rotary drum filling techniquescommonly known in the art. Such processes may be either continuous orintermittent. The film may be dampened, and/or heated to increase themalleability thereof. The method may also involve the use of a vacuum todraw the film into a suitable mold. The vacuum drawing the film into themold can be applied for about 0.2 to about 5 seconds, or about 0.3 toabout 3, or about 0.5 to about 1.5 seconds, once the film is on thehorizontal portion of the surface. This vacuum can be such that itprovides an under-pressure in a range of 10 mbar to 1000 mbar, or in arange of 100 mbar to 600 mbar, for example.

The molds, in which packets may be made, can have any shape, length,width and depth, depending on the required dimensions of the pouches.The molds may also vary in size and shape from one to another, ifdesirable. For example, the volume of the final pouches may be about 5mL to about 300 mL, or about 10 mL to 150 mL, or about 20 mL to about100 mL, and that the mold sizes are adjusted accordingly.

In one aspect, the packet includes a first and a second sealedcompartment. The second compartment is in a generally superposedrelationship with the first sealed compartment such that the secondsealed compartment and the first sealed compartment share a partitioningwall interior to the pouch.

In one aspect, the packet including a first and a second compartmentfurther includes a third sealed compartment. The third sealedcompartment is in a generally superposed relationship with the firstsealed compartment such that the third sealed compartment and the firstsealed compartment share a partitioning wall interior to the pouch.

In various aspects, the first composition and the second composition areselected from one of the following combinations: liquid, liquid; liquid,powder; powder, powder; and powder, liquid.

In various aspects, the first, second and third compositions areselected from one of the following combinations: solid, liquid, liquidand liquid, liquid, liquid.

In one aspect, the single compartment or plurality of sealedcompartments contains a composition. The plurality of compartments mayeach contain the same or a different composition. The composition isselected from a liquid, solid or combination thereof.

Heat can be applied to the film in the process commonly known asthermoforming. The heat may be applied using any suitable means. Forexample, the film may be heated directly by passing it under a heatingelement or through hot air, prior to feeding it onto a surface or onceon a surface. Alternatively, it may be heated indirectly, for example byheating the surface or applying a hot item onto the film. The film canbe heated using an infrared light. The film may be heated to atemperature of at least 50° C., for example about 50 to about 150° C.,about 50 to about 120° C., about 60 to about 130° C., about 70 to about120° C., or about 60 to about 90° C.

Alternatively, the film can be wetted by any suitable means, for exampledirectly by spraying a wetting agent (including water, a solution of thefilm composition, a plasticizer for the film composition, or anycombination of the foregoing) onto the film, prior to feeding it ontothe surface or once on the surface, or indirectly by wetting the surfaceor by applying a wet item onto the film.

Once a film has been heated and/or wetted, it may be drawn into anappropriate mold, preferably using a vacuum. The film can bethermoformed with a draw ratio of at least about 1.5, for example, andoptionally up to a draw ratio of 2, for example. The filling of themolded film can be accomplished by utilizing any suitable means. In someembodiments, the most preferred method will depend on the product formand required speed of filling. In some embodiments, the molded film isfilled by in-line filling techniques. The filled, open packets are thenclosed forming the pouches, using a second film, by any suitable method.This may be accomplished while in horizontal position and in continuous,constant motion. The closing may be accomplished by continuously feedinga second film, preferably water-soluble film, over and onto the openpackets and then preferably sealing the first and second film together,typically in the area between the molds and thus between the packets.

Any suitable method of sealing the packet and/or the individualcompartments thereof may be utilized. Non-limiting examples of suchmeans include heat sealing, solvent welding, solvent or wet sealing, andcombinations thereof. The water-soluble packet and/or the individualcompartments thereof can be heat sealed at a temperature of at least200° F. (93° C.), for example in a range of about 220° F. (about 105°C.) to about 290° F. (about 145° C.), or about 230° F. (about 110° C.)to about 280° F. (about 140° C.). Typically, only the area which is toform the seal is treated with heat or solvent. The heat or solvent canbe applied by any method, typically on the closing material, andtypically only on the areas which are to form the seal. If solvent orwet sealing or welding is used, it may be preferred that heat is alsoapplied. Preferred wet or solvent sealing/welding methods includeselectively applying solvent onto the area between the molds, or on theclosing material, by for example, spraying or printing this onto theseareas, and then applying pressure onto these areas, to form the seal.Sealing rolls and belts as described above (optionally also providingheat) can be used, for example.

The formed pouches may then be cut by a cutting device. Cutting can beaccomplished using any known method. It may be preferred that thecutting is also done in continuous manner, and preferably with constantspeed and preferably while in horizontal position. The cutting devicecan, for example, be a sharp item, or a hot item, or a laser, whereby inthe latter cases, the hot item or laser ‘burns’ through the film/sealingarea.

The different compartments of a multi-compartment pouches may be madetogether in a side-by-side style wherein the resulting, cojoined pouchesmay or may not be separated by cutting. Alternatively, the compartmentscan be made separately.

In some embodiments, pouches may be made according to a processincluding the steps of:

-   -   a) forming a first compartment (as described above);    -   b) forming a recess within some or all of the closed compartment        formed in step (a), to generate a second molded compartment        superposed above the first compartment;    -   c) filling and closing the second compartments by means of a        third film;    -   d) sealing the first, second and third films; and    -   e) cutting the films to produce a multi-compartment pouch.

The recess formed in step (b) may be achieved by applying a vacuum tothe compartment prepared in step (a).

In some embodiments, second, and/or third compartment(s) can be made ina separate step and then combined with the first compartment asdescribed in EP 2088187 or WO 2009/152031.

In other embodiments, pouches may be made according to a processincluding the steps of:

-   -   a) forming a first compartment, optionally using heat and/or        vacuum, using a first film on a first forming machine;    -   b) filling the first compartment with a first composition;    -   c) on a second forming machine, deforming a second film,        optionally using heat and vacuum, to make a second and        optionally third molded compartment;    -   d) filling the second and optionally third compartments;    -   e) sealing the second and optionally third compartment using a        third film;    -   f) placing the sealed second and optionally third compartments        onto the first compartment;    -   g) sealing the first, second and optionally third compartments;        and    -   h) cutting the films to produce a multi-compartment pouch.

The first and second forming machines may be selected based on theirsuitability to perform the above process. In some embodiments, the firstforming machine is preferably a horizontal forming machine, and thesecond forming machine is preferably a rotary drum forming machine,preferably located above the first forming machine.

It should be understood that by the use of appropriate feed stations, itmay be possible to manufacture multi-compartment pouches incorporating anumber of different or distinctive compositions and/or different ordistinctive liquid, gel or paste compositions.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US 2003/0087791; US 20030087790; US20050003983A1; US 2004/0048764; U.S. Pat. Nos. 4,762,636; 6,291,412; US2005/0227891; EP 1070115; U.S. Pat. Nos. 5,879,584; 5,691,297;5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; 5,486,303 all ofwhich are incorporated herein by reference. The compositions of theinvention or prepared according to the invention comprise cleaningand/or treatment composition including, but not limited to, compositionsfor treating fabrics, hard surfaces and any other surfaces in the areaof fabric and home care, including: air care including air freshenersand scent delivery systems, car care, dishwashing, fabric conditioning(including softening and/or freshening), laundry detergency, laundry andrinse additive and/or care, hard surface cleaning and/or treatmentincluding floor and toilet bowl cleaners, granular or powder-formall-purpose or “heavy-duty” washing agents, especially cleaningdetergents; liquid, gel or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid types; liquid fine-fabricdetergents; hand dishwashing agents or light duty dishwashing agents,especially those of the high-foaming type; machine dishwashing agents,including the various tablet, granular, liquid and rinse-aid types forhousehold and institutional use: car or carpet shampoos, bathroomcleaners including toilet bowl cleaners; as well as cleaning auxiliariessuch as bleach additives and “stain-stick” or pre-treat types,substrate-laden compositions such as dryer added sheets. Preferred arecompositions and methods for cleaning and/or treating textiles and/orhard surfaces, most preferably textiles. The compositions are preferablycompositions used in a pre-treatment step or main wash step of a washingprocess, most preferably for use in textile washing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Uses

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. It is contemplated that cleaning asdescribed may be both in small scale as in, e.g., family house hold aswell as in large scale as in, e.g., industrial and professionalsettings. In one aspect of the invention, the method comprises the stepof contacting the surface to be treated in a pre-treatment step or mainwash step of a washing process, most preferably for use in a textilewashing step or alternatively for use in dishwashing including bothmanual as well as automated/mechanical dishwashing. In one aspect of theinvention the lipase variant and other components are added sequentiallyinto the method for cleaning and/or treating the surface. Alternatively,the lipase variant and other components are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO 2008/101958 and/or by applying alternating pressure(pressure/vaccum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one aspect of Applicants' cleaning composition, cleaning additiveor mixture thereof. The fabric may comprise most any fabric capable ofbeing laundered in normal consumer or institutional use conditions. Thesolution may have a pH from 8 to 10.5. The compositions may be employedat concentrations from 500 to 15.000 ppm in solution. The watertemperatures typically range from 5° C. to 90° C. The water to fabricratio is typically from 1:1 to 30:1.

The invention also provides methods for hydrolyzing a lipase substratecomprising mixing the substrate with a lipase variant of the inventionat conditions conductive for the lipase variant hydrolyzing thesubstrate.

In one aspect the invention relates to a method for hydrolyzing a lipasesubstrate comprising: Adding to said substrate a lipase variant of aparent lipase, which variant comprises a substitution at one or morepositions corresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V,A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the mature polypeptide of the parentlipase, has lipase activity, and has at least 60% but less than 100%sequence identity to the parent lipase or a fragment thereof with lipaseactivity, in which method odor generation is reduced when compared tothe method wherein the parent lipase is added to the lipase substrate.In one aspect the parent lipase comprises or consists of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8,SEQ ID NO: 10, or SEQ ID NO: 12.

The invention also provides methods for removing lipid stain materialfrom a surface comprising contacting the lipid stain material with alipase variant of the invention or a composition of the invention atconditions conductive for he lipase variant hydrolyzing the lipid stainmaterial. In one aspect the invention relates to a method for lipidstain removal from a surface comprising: Contacting said stain with alipase variant of a parent lipase, which variant comprises asubstitution at one or more positions corresponding to positionsF7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase, has lipase activity, andhas at least 60% but less than 100% sequence identity to the parentlipase, in which method odor generation is reduced when compared to themethod wherein the parent lipase is added to the lipase substrate. Inone aspect the parent lipase comprises or consists of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8,SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof with lipaseactivity.

The invention also provides use of a lipase variant of the invention orthe composition of the invention for cleaning a surface comprisingapplying the lipase variant to the surface to be cleaned.

In one aspect the invention relates to use of a lipase variant of aparent lipase for cleaning a surface comprising: applying to saidsurface to be cleaned a lipase variant of a parent lipase, which variantcomprises a substitution at one or more positions corresponding topositions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase, has lipase activity, andhas at least 60% but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or a fragment thereof with lipase activity,in which use odor generation is reduced when compared to the use whereinthe parent lipase is added to the surface to be cleaned. In one aspectthe parent lipase comprises or consists of the mature polypeptide of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQID NO: 12, or a fragment thereof with lipase activity.

In one aspect the invention relates to a method of producing thecomposition, comprising adding a lipase variant of a parent lipase,wherein the variant comprises a substitution at one or more positionscorresponding to positions F7H/K/R, F51A/I/L/V/Y, T143A/G/S/V, A150G/V,H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, P256A/K/N/Q/R/S/T/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y of the parent lipase has lipase activity, andhas at least 60% but less than 100% sequence identity to the parentlipase, in which method odor generation is reduced when compared to themethod wherein the parent lipase is added to the lipase substrate. Inone aspect the invention relates to a method for hydrolyzing a lipidpresent in a soil and/or a stain on a surface, comprising contacting thesoil and/or the stain with the cleaning composition. In one aspect theinvention relates to use of the composition in the hydrolysis of acarboxylic acid ester. In one aspect the invention relates to use of thecomposition in the hydrolysis, synthesis or interesterification of anester. In one aspect the invention relates to use of the composition forthe manufacture of a low odor composition. In one aspect the inventionrelates to use of the composition for the manufacture of a low odorformulation. In one aspect the parent lipase comprises or consists ofthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof withlipase activity.

EXAMPLES Example 1—Assays

p-Nitrophenyl (pNP) Assay:

The hydrolytic activity of a lipase may be determined by a kinetic assayusing p-nitrophenyl acyl esters as substrate.

A 100 mM stock solution in DMSO of the substrates: p-Nitrophenylbutyrate (C4), p-Nitrophenyl caproate (C6), p-Nitrophenyl caprate (C10),p-Nitrophenyl laurate (C12) and p-Nitrophenyl palmitate (C16) (all fromSigma-Aldrich Danmark A/S, Kirkebjerg Allé 84, 2605 Brøndby; Cat. no.:C4:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) may bediluted to a final concentration of 1 mM 25 into assay buffer (50 mMTris; pH 7.7; 0.4% TritonX-100).

The lipase of the invention, the parent lipase and appropriate controls,e.g., Buffer (negative), Lipolase™ & Lipex™ (positive) in 50 mM Hepes;pH 8.0; 10 ppm TritonX-100; +/−20 mM CaCl₂ may be added to the substratesolution in the following final concentrations: 0.01 mg/ml; 5×10-3mg/ml; 2.5×10-4 mg/ml; and 1.25×10-4 mg/ml in 96-well NUNC plates (Cat.No: 260836, Kamstrupvej 90, DK-4000, Roskilde). Release of p-nitrophenolby hydrolysis of p-nitrophenyl acyl may be monitored at 405 nm for 5minutes in 10 second intervals on a Spectra max 190 (Molecular DevicesGmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY). Thehydrolytic activity towards one or more substrates of a variant may becompared to that of the parent lipase.

Relative Wash Performance (RP(Wash))

Washing experiments were performed using Automatic Mechanical StressAssay (AMSA) in order to assess the wash performance in laundry. TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid are vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO 02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted under the experimental conditionsspecified below:

Detergent: 3.3 g/L Detergent B Test solution volume: 160 uL Wash time:20 minutes Temperature: 30° C. Lipase dosage: 0 ppm or 0.35 ppm Testmaterial: Cream Annatto stained EMPA221 cotton textile prepared asdescribed in WO 2006/125437 except to exchanging turmeric with annatto(Annatto: A-320-WS, Chr. Hansen A/S, Boege Alle{acute over ( )} 10-12,DK-2970, Hoersholm, Denmark & EMPA221: EMPA, Lerchenfeldstrasse 5,CH-9014, St. Gallen, Switzerland) Water hardness was adjusted to 15° dHby addition of CaCl₂, MgCl₂ and NaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻ = 4:1:7.5).

Composition Detergent B wt % NaOH, pellets (>99%) 1.05 Linearalkylbenzenesulfonic acid (LAS) (97%) 7.20 Sodium laureth sulfate (SLES)(28%) 10.58 Soy fatty acid (>90%) 2.75 Coco fatty acid (>99%) 2.75Alcohol ethoxylate (AEO) with 8 mol EO; Lutensol TO 8 6.60 (~100%)Triethanol amine (100%) 3.33 Na-citrate, dihydrate (100%) 2.00 DTMPA;0.48 diethylenetriaminepentakis(methylene)pentakis(phosphonic acid),heptasodium salt (Dequest 2066 C) (~42% as Na7 salt) MPG (>98%) 6.00EtOH, propan-2-ol (90/10%) 3.00 Glycerol (>99.5) 1.71 Sodium formate(>95%) 1.00 PCA (40% as sodium salt) 0.46 Water up to 100

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 100° C. for 15 minutes.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with annatto. Annatto containsthe colorant norbixin, which function as a pH indicator by having pHdependent color change. Lipase activity leads to release of free fattyacids from the cream acylglycerols and this leads to pH decrease andthereby color change of the norbixin pH indicator. Lipase washperformance can therefore be expressed as the extent of color change oflight reflected-emitted from the washed soiled textile when illuminatedwith white light.

Color measurements were made with a professional flatbed scanner (EPSONEXPRESSION 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark),which was used to capture an image of the washed soiled textile. Toextract a value for the light intensity from the scanned images, 24-bitpixel values from the image were converted into values for red, greenand blue (RGB).

Color change due to lipase activity was measured as the change in thereflection-emitting of green light (G) relative to the light intensityvalue (Int) calculated as:Int=√{square root over (R²+G²+B²)}

The relative wash performance (RP(Wash)) of a lipase relative to areference lipase was calculated as:RP(Wash)=(G/Int(variant)−G/Int(no enzyme))/(G/Int(lipase ref.)−G/Int(noenzyme)).

A lipase is considered to exhibit improved wash performance, if itperforms better than the reference (RP(Wash)>1). In the context of thepresent invention the reference enzyme is a lipase having a single aminoacid substitution relative to the tested lipase.

Odor Detection by Solid Phase Micro Extraction Gas ChromatographMeasurements.

The butyric acid release (odor) from the lipase washed swatches weremeasured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC)using the following method.

Cream Annatto stained EMPA221 cotton textile was washed as specifiedabove and after wash, excess water was removed from the textile usingfilter paper and the textile was thereafter dried at 25° C. for 2 h.Each SPME-GC measurement was performed with four pieces of the washedand dried textile (5 mm in diameter), which were transferred to a GasChromatograph (GC) vial and the vial was closed. The samples wereincubated at 30° C. for 24 hours and subsequently heated to 140° C. for30 minutes and stored at 20° C.-25° C. for at least 4 hours beforeanalysis. The analyses were performed on a Varian 3800 GC equipped witha Stabilwax-DA w/Integra-Guard column (30 m, 0.32 mm ID and 0.25 um df)and a Carboxen PDMS SPME fiber (85 micro-m). Sampling from each GC vialwas done at 50° C. for 8 minutes with the SPME fiber in the head-spaceover the textile pieces and the sampled compounds were subsequentlyinjected onto the column (injector temperature=250° C.). Column flow=2ml helium/minute. Column oven temperature gradient: 0 minute=50° C., 2minutes=50° C., 6 minutes 45 seconds=240° C., 11 minutes 45 seconds=240°C. Detection was done using a Flame Ionization Detector (FID) and theretention time for butyric acid was identified using an authenticstandard.

The relative odor release (RP(Odor)) of a lipase is the ratio betweenthe amount butyric acid released (peak area) from a lipase washed swatchand the amount butyric acid released (peak area) from a reference lipasewashed swatch, after both values have been corrected for the amount ofbutyric acid released (peak area) from a non-lipase washed swatch(blank). The reference lipase is a polypeptide having a single aminoacid substitution relative to the tested lipase. The relative odorperformance (RP(Odor)) of the polypeptide is calculated in accordancewith the below formula:RP(Odor)=(odor(variant)−odor(no enzyme))/(odor(lipase ref.)−odor(noenzyme))Where odor is the measured butyric acid (peak area) released from thetextile surface.Benefit Risk Factor (RP(Wash)/RP(Odor))

The Benefit Risk factor describing the wash performance (Benefit)compared to the odor release (Risk) can be defined as RP(Wash)/RP(Odor).If the Benefit Risk factor of a lipase is higher than 1, the lipase hasbetter wash performance relative to the released odor compared to thereference lipase. Each of the result tables below represent a separateexperiment using the the reference lipase in the top most row designated“(reference lipase)” and setting the RP (wash), the RP(odor) and theRP(wash/Rp(odor) to 1 for the reference lipase. Performance of theremaining lipase variant in the separate experiment is then calculatedrelatively to the reference lipase for that experiment.

Example 2—Results

Substitution in SEQ ID NO: 2 RP (Wash) RP (odor) RP(Wash)/RP(odor) —(reference lipase) 1.00 1.00 1.00 F7K 0.98 0.71 1.38 F51L 1.04 0.78 1.35F51V 0.84 0.65 1.28 F51Y 0.94 0.72 1.31 H198D 1.10 0.71 1.53 H198G 0.960.48 2.00 H198F 0.82 0.30 2.75 H198I 0.91 0.31 2.94 H198N 0.99 0.51 1.93H198S 0.96 0.54 1.78 H198T 0.92 0.41 2.25 H198Y 0.95 0.34 2.80 N200Q0.85 0.59 1.44 S224F 1.19 0.91 1.33 L227D 0.83 0.61 1.36 L227E 0.87 0.741.17 L227R 0.97 0.80 1.21 V228P 1.20 0.88 1.39 V230R 1.31 1.08 1.21I255G 0.91 0.53 1.73 I255N 0.84 0.45 1.87 A257F 0.96 0.52 1.84 A257I0.89 0.29 3.09 I255T A257V 0.86 0.36 2.40 V230L L259F W260H 0.86 0.441.952.1

Substitution in SEQ ID NO: 2 RP (Wash) RP (odor) RP(Wash)/RP(odor) N33KK98E (reference lipase) 1.00 1.00 1.00 N33K F51V K98E 1.04 0.78 1.34N33K F51V K98E N101D 1.00 1.00 1.00 (reference lipase) N33K F51V K98EN101D H198S 0.95 0.52 1.81 N33K F51V E56K L69R K98E 1.00 1.00 1.00(reference lipase) N33K F51V E56K L69R K98E H198I 0.73 0.30 2.47 N33KF51V E56K L69R K98E H198L 0.73 0.29 2.53 N33K F51V E56K L69R K98E H198N0.89 0.55 1.61 N33K F51V E56K L69R K98E H198S 0.84 0.52 1.62 N33K F51VE56K L69R K98E H198Y 0.86 0.34 2.51 — (reference lipase) 1.00 1.00 1.00F51I 1.14 0.91 1.25 H198L 0.98 0.40 2.44 S224P 1.08 0.89 1.21 V2K N33KF51V (reference lipase) 1.00 1.00 1.00 V2K N33K F51V H198N 0.91 0.551.68 V2K N33K F51V H198S 0.92 0.55 1.65 V2K N33K F51V S224F 0.95 0.741.28 V2K N33K F51V S224I 0.90 0.75 1.20 V2K N33K F51V V228P 0.89 0.731.21 V2K N33K F51V V230R 1.04 0.67 1.56 V2K N33K F51V A257V 0.85 0.392.18 N33K F51V E56K L69R K98E 1.00 1.00 1.00 (reference lipase) N33KF51V E56K L69R K98E A257I 0.74 0.24 3.06 N33K F51V E56K L69R K98E A257V0.83 0.43 1.96 V2K N33K F51V L69R K98E V176L E210K 1.00 1.00 1.00 L227G(reference lipase) V2K N33K F51V L69R K98E V176L H198S 0.87 0.61 1.42E210K L227G V2K N33K F51V L69R K98E V176L E210K 0.89 0.66 1.35 S224FL227G V2K N33K F51V L69R K98E V176L E210K 0.73 0.44 1.64 L227G A257VN33K F51V S54T E56K K98I 1.00 1.00 1.00 (reference lipase) N33K F51VS54T E56K K98I T143A 0.94 0.77 1.22 N33K F51V S54T E56K K98I H198N 0.830.44 1.89 N33K F51V S54T E56K K98I H198S 0.79 0.47 1.68 N33K F51V S54TE56K K98I H198Y 0.79 0.26 2.99 N33K F51V S54T E56K K98I A257I 0.98 0.332.94 N33K F51L S54T E56K K98I 1.00 1.00 1.00 (reference lipase) N33KF51L S54T E56K K98I A257I 0.75 0.24 3.13 N33K F51V K98E N101D E210K 1.001.00 1.00 (reference lipase) N33K F51V K98E N101D H198S E210K S224P 0.750.40 1.88 AMSA AMSA AMSA RP(Wash)/ Substitution in SEQ ID NO: 10RP(Wash) RP(odor) RP(odor) D27R G38A G91T D96E D111A G163K T231R 1.001.00 1.00 N233R D254S (reference lipase) D27R G38A G91T D96E D111A G163KT231R 0.93 0.78 1.20 N233R D254S P256T N33Q D27R G38A G91T D96E D111AG163K 1.00 1.00 1.00 T231R N233R D254S (reference lipase) N33Q D27R G38AG91T D96E D111A G163K 1.02 0.72 1.40 T231R N233R D254S P256T

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A lipase variant having lipase activity andhaving between 80% to less than 100% sequence identity to a parentlipase comprising the amino acid sequence of SEQ ID NO: 2, wherein thevariant comprises one or more substitutions selected from A257I/F/V,F7K, S224F/P, and V230R using SEQ ID NO:2 for position numbering.
 2. Thelipase variant of claim 1, wherein the variant comprises thesubstitutions F51A/I/L/V/Y and T256A/K/N/Q/R/S/P/W and one or moresubstitutions selected from H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, F7H/K/R,T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y,L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y,A257F/H/l/L/V/Y, L259F/Y, and W260D/E/F/H/l/L/N/Q/S/T/Y, using SEQ IDNO: 2 for position numbering.
 3. The lipase variant of claim 1, whereinthe variant comprises the substitution A257I and one or moresubstitutions selected from F51A/I/L/V/Y, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,F7H/K/R, T143A/G/S/V, A150G/V, N200H/K/Q/R, I202G/L/V,S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R, V230H/K/L/R,I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y, exhibiting both a reduced odor generationupon hydrolyzing a lipase substrate and an improved stability comparedto the parent lipase.
 4. The lipase variant of claim 1, wherein thevariant comprises the substitutions F51A/I/L/V/Y and/orH198A/D/E/F/G/I/L/N/Q/S/T/V/Y, using SEQ ID NO:2 for position numbering.5. The lipase variant of claim 1, wherein the variant comprises thesubstitution T256A/K/N/Q/R/S/P/W and one or more substitutions selectedfrom F7H/K/R, T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,N200H/K/Q/R, I202G/LN, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, A257F/H/I/L/V/Y, L259F/Y, andW260D/E/F/H/I/L/N/Q/S/T/Y, using SEQ ID NO:2 for position numbering. 6.A lipase variant of claim 1, comprising a substitution at one or morepositions corresponding to positions: F7H/K/R, F51A/I/L/V/Y,T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y, N200H/K/Q/R,I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P, P229H/K/R,V230H/K/L/R, I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W, A257F/H/I/L/V/Y,L259F/Y, and W260D/E/F/H/l/L/N/Q/S/T/Y of the parent lipase, wherein thevariant has lipase activity and has at least 80% but less than 100%sequence identity to the parent lipase.
 7. The variant of claim 1,wherein the variant has at least 85% sequence identity to the parentlipase comprising SEQ ID NO:
 2. 8. The variant of claim 1, wherein thevariant has at least 90% sequence identity to the parent lipasecomprising SEQ ID NO:
 2. 9. The variant of claim 1, which furthercomprises a substitution at one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or each positions corresponding to any of positions 7, 51, 143, 150,198, 200, 202, 224, 227, 228, 229, 230, 255, 256, 257, 259, 260, usingSEQ ID NO: 2 for position numbering.
 10. The variant of claim 1, whichfurther comprises one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or eachsubstitutions selected from the group consisting of: F7H/K/R,F51A/I/L/V/Y, T143A/G/S/V, A150G/V, H198A/D/E/F/G/I/L/N/Q/S/T/V/Y,N200H/K/Q/R, I202G/L/V, S224C/F/H/I/L/P/Y, L227D/E/K/R, V228P,P229H/K/R, V230H/K/L/R, I255A/G/N/P/S/T/V/Y, T256A/K/N/Q/R/S/P/W,L259F/Y, and W260D/E/F/H/I/L/N/Q/S/T/Y.
 11. A composition comprising thevariant of claim
 1. 12. The composition of claim 11, wherein thecomposition is a detergent composition.
 13. A polynucleotide encodingthe variant of claim
 1. 14. A microbial host cell comprising thepolynucleotide of claim
 13. 15. A method of producing a lipase variantof a parent lipase, comprising: (a) cultivating the host cell of claim14 under conditions suitable for expression of the variant; and (b)recovering the variant.
 16. A method for removing lipid stain materialfrom a surface, comprising contacting the lipid stain material with alipase variant of claim 1 at conditions conducive for the lipase variantto hydrolyze the lipid stain material.
 17. The variant of claim 1,further comprising substitutions selected from: 1) F7H; 2) F7R; 3) F51A;4) F51I; 5) F51L; 6) F51V; 7) F51Y; 8) T143A; 9) T143G; 10) T143S; 11)T143V; 12) A150G; 13) A150V; 14) H198A; 15) H198D; 11) H198E; 12) H198F;13) H198G; 19) H198I; 20) H198L; 14) H198N; 15) H198Q; 23) H198S; 24)H198T; 25) H198V; 26) H198Y; 16) N200H; 28) N200K; 29) N200Q; 30) N200R;31) I202G; 32) I202L; 17) I202V; 34) S224C; 35) S224H; 36) S224I; 37)S224L; 38) S224Y; 39) L227D; 40) L227E; 41) L227K; 42) L227R; 43) V228P;44) P229H; 45) P229K; 46) P229R; 47) V230H; 48) V230K; 49) V230L; 50)I255A; 51) I255G; 52) I255N; 53) I255P; 54) I255S; 55) I255T; 56) I255V;57) I255Y; 58) P256A; 59) P256K; 60) P256N; 61) P256Q; 62) P256R; 63)P256S; 64) P256T; 65) P256W; 66) T256A; 67) T256K; 68) T256N; 69) T256Q;70) T256R; 71) T256S; 72) T256P; 73) T256W; 74) A257H; 75) A257L; 76)A257Y; 77) L259F; 78) L259Y; 79) W260D; 80) W260E; 81) W260F; 82) W260H;83) W260I; 84) W260L; 85) W260N; 86) W260Q; 87) W260S; 88) W260T; 89)W260Y; 90) E1C V2K N33K F51I T143A R233C; 91) E1C V2K N33K F51I A150GR233C; 92) E1C V2K N33K F51I H198I R233C; 93) E1C V2K N33K F51I H198LR233C; 94) E1C V2K N33K F51I H198N R233C; 95) E1C V2K N33K F51I H198SR233C; 96) E1C V2K N33K F51I H198Y R233C; 97) E1C V2K N33K F51I S224IR233C; 98) E1C V2K N33K F51I L227D R233C; 99) E1C V2K N33K F51I V228PR233C; 100) E1C V2K N33K F51I R233C I255G; 101) E1C V2K N33K F51L T143AR233C; 102) E1C V2K N33K F51L A150G R233C; 103) E1C V2K N33K F51L H198IR233C; 104) E1C V2K N33K F51L H198L R233C; 105) E1C V2K N33K F51L H198NR233C; 106) E1C V2K N33K F51L H198S R233C; 107) E1C V2K N33K F51L H198YR233C; 108) E1C V2K N33K F51L S224I R233C; 109) E1C V2K N33K F51L L227DR233C; 110) E1C V2K N33K F51L V228P R233C; 111) E1C V2K N33K F51L R233CI255G; 112) E1C V2K N33K F51V T143A R233C; 113) E1C V2K N33K F51V A150GR233C; 114) E1C V2K N33K F51V H198I R233C; 115) E1C V2K N33K F51V H198LR233C; 116) E1C V2K N33K F51V H198N R233C; 117) E1C V2K N33K F51V H198SR233C; 118) E1C V2K N33K F51V H198Y R233C; 119) E1C V2K N33K F51V S224IR233C; 120) E1C V2K N33K F51V L227D R233C; 121) E1C V2K N33K F51V V228PR233C; 122) E1C V2K N33K F51V R233C I255G; 123) E1C V2Y N33K F51I T143AR233C; 124) E1C V2Y N33K F51I A150G R233C; 125) E1C V2Y N33K F51I H198IR233C; 126) E1C V2Y N33K F51I H198L R233C; 127) E1C V2Y N33K F51I H198NR233C; 128) E1C V2Y N33K F51I H198S R233C; 129) E1C V2Y N33K F51I H198YR233C; 130) E1C V2Y N33K F51I S224I R233C; 131) E1C V2Y N33K F51I L227DR233C; 132) E1C V2Y N33K F51I V228P R233C; 133) E1C V2Y N33K F51I R233CI255G; 134) E1C V2Y N33K F51L T143A R233C; 135) E1C V2Y N33K F51L A150GR233C; 136) E1C V2Y N33K F51L H198I R233C; 137) E1C V2Y N33K F51L H198LR233C; 138) E1C V2Y N33K F51L H198N R233C; 139) E1C V2Y N33K F51L H198SR233C; 140) E1C V2Y N33K F51L H198Y R233C; 141) E1C V2Y N33K F51L S224IR233C; 142) E1C V2Y N33K F51L L227D R233C; 143) E1C V2Y N33K F51L V228PR233C; 144) E1C V2Y N33K F51L R233C I255G; 145) E1C V2Y N33K F51V T143AR233C; 146) E1C V2Y N33K F51V A150G R233C; 147) E1C V2Y N33K F51V H198IR233C; 148) E1C V2Y N33K F51V H198L R233C; 149) E1C V2Y N33K F51V H198NR233C; 150) E1C V2Y N33K F51V H198S R233C; 151) E1C V2Y N33K F51V H198YR233C; 152) E1C V2Y N33K F51V S224I R233C; 153) E1C V2Y N33K F51V L227DR233C; 154) E1C V2Y N33K F51V V228P R233C; 155) E1C V2Y N33K F51V R233CI255G; 156) V2K N33K F51I L69R K98E T143A V176L E210K L227G; 157) V2KN33K F51I L69R K98E A150G V176L E210K L227G; 158) V2K N33K F51I L69RK98E V176L H198I E210K L227G; 159) V2K N33K F51I L69R K98E V176L H198LE210K L227G; 160) V2K N33K F51I L69R K98E V176L H198N E210K L227G; 161)V2K N33K F51I L69R K98E V176L H198S E210K L227G; 162) V2K N33K F51I L69RK98E V176L H198Y E210K L227G; 163) V2K N33K F51I L69R K98E V176L E210KS224I L227G; 164) V2K N33K F51I L69R K98E V176L E210K L227D; 165) V2KN33K F51I L69R K98E V176L E210K L227G I255G; 166) V2K N33K F51I L69RK98E V176L E210K L227G V228P; 167) V2K N33K F51I T143A; 168) V2K N33KF51I A150G 169) V2K N33K F51I H198I; 170) V2K N33K F51I H198L; 171) V2KN33K F51I H198N; 172) V2K N33K F51I H198S; 173) V2K N33K F51I H198Y;174) V2K N33K F51I S224I; 175) V2K N33K F51I L227D; 176) V2K N33K F51IV228P; 177) V2K N33K F51I I255G; 178) V2K N33K F51L L69R K98E T143AV176L E210K L227G; 179) V2K N33K F51L L69R K98E A150G V176L E210K L227G;180) V2K N33K F51L L69R K98E V176L H198I E210K L227G; 181) V2K N33K F51LL69R K98E V176L H198L E210K L227G; 182) V2K N33K F51L L69R K98E V176LH198N E210K L227G; 183) V2K N33K F51L L69R K98E V176L H198S E210K L227G;184) V2K N33K F51L L69R K98E V176L H198Y E210K L227G; 185) V2K N33K F51LL69R K98E V176L E210K S224I L227G; 186) V2K N33K F51L L69R K98E V176LE210K L227D; 187) V2K N33K F51L L69R K98E V176L E210K L227G I255G; 188)V2K N33K F51L L69R K98E V176L E210K L227G V228P; 189) V2K N33K F51LT143A; 190) V2K N33K F51L A150G; 191) V2K N33K F51L H198I; 192) V2K N33KF51L H198L; 193) V2K N33K F51L H198N; 194) V2K N33K F51L H198S; 195) V2KN33K F51L H198Y; 196) V2K N33K F51L S224I; 197) V2K N33K F51L L227D;198) V2K N33K F51L V228P; 199) V2K N33K F51L I255G; 200) V2K N33K F51VL69R K98E T143A V176L E210K L227G; 201) V2K N33K F51V L69R K98E A150GV176L E210K L227G; 202) V2K N33K F51V L69R K98E V176L H198I E210K L227G;203) V2K N33K F51V L69R K98E V176L H198L E210K L227G; 204) V2K N33K F51VL69R K98E V176L H198N E210K L227G; 205) V2K N33K F51V L69R K98E V176LH198S E210K L227G; 206) V2K N33K F51V L69R K98E V176L H198Y E210K L227G;207) V2K N33K F51V L69R K98E V176L E210K S224I L227G; 208) V2K N33K F51VL69R K98E V176L E210K L227D; 209) V2K N33K F51V L69R K98E V176L E210KL227G V228P; 210) V2K N33K F51V L69R K98E V176L E210K L227G I255G; 211)V2K N33K F51V T143A; 212) V2K N33K F51V A150G; 213) V2K N33K F51V H198I;214) V2K N33K F51V H198L; 215) V2K N33K F51V H198N; 216) V2K N33K F51VH198S; 217) V2K N33K F51V H198Y; 218) V2K N33K F51V S224I; 219) V2K N33KF51V L227D; 220) V2K N33K F51V V228P; 221) V2K N33K F51V I255G; 222) V2YN33K F51I L69R K98E T143A V176L E210K L227G; 223) V2Y N33K F51I L69RK98E A150G V176L E210K L227G; 224) V2Y N33K F51I L69R K98E V176L H198IE210K L227G; 225) V2Y N33K F51I L69R K98E V176L H198L E210K L227G; 226)V2Y N33K F51I L69R K98E V176L H198N E210K L227G; 227) V2Y N33K F51I L69RK98E V176L H198S E210K L227G; 228) V2Y N33K F51I L69R K98E V176L H198YE210K L227G; 229) V2Y N33K F51I L69R K98E V176L E210K S224I L227G; 230)V2Y N33K F51I L69R K98E V176L E210K L227D; 231) V2Y N33K F51I L69R K98EV176L E210K L227G V228P; 232) V2Y N33K F51I L69R K98E V176L E210K L227GI255G; 233) V2Y N33K F51I T143A; 234) V2Y N33K F51I A150G 235) V2Y N33KF51I H198I; 236) V2Y N33K F51I H198L; 237) V2Y N33K F51I H198N; 238) V2YN33K F51I H198S; 239) V2Y N33K F51I H198Y; 240) V2Y N33K F51I S224I;241) V2Y N33K F51I L227D; 242) V2Y N33K F51I V228P; 243) V2Y N33K F51II255G; 244) V2Y N33K F51L L69R K98E T143A V176L E210K L227G; 245) V2YN33K F51L L69R K98E A150G V176L E210K L227G; 246) V2Y N33K F51L L69RK98E V176L H198I E210K L227G; 247) V2Y N33K F51L L69R K98E V176L H198LE210K L227G; 248) V2Y N33K F51L L69R K98E V176L H198N E210K L227G; 249)V2Y N33K F51L L69R K98E V176L H198S E210K L227G; 250) V2Y N33K F51L L69RK98E V176L H198Y E210K L227G; 251) V2Y N33K F51L L69R K98E V176L E210KS224I L227G; 252) V2Y N33K F51L L69R K98E V176L E210K L227D; 253) V2YN33K F51L L69R K98E V176L E210K L227G V228P; 254) V2Y N33K F51L L69RK98E V176L E210K L227G I255G; 255) V2Y N33K F51L T143A; 256) V2Y N33KF51L A150G 257) V2Y N33K F51L H198I; 258) V2Y N33K F51L H198L; 259) V2YN33K F51L H198N; 260) V2Y N33K F51L H198S; 261) V2Y N33K F51L H198Y;262) V2Y N33K F51L S224I; 263) V2Y N33K F51L L227D; 264) V2Y N33K F51LV228P; 265) V2Y N33K F51L I255G; 266) V2Y N33K F51V L69R K98E T143AV176L E210K L227G; 267) V2Y N33K F51V L69R K98E A150G V176L E210K L227G;268) V2Y N33K F51V L69R K98E V176L H198I E210K L227G; 269) V2Y N33K F51VL69R K98E V176L H198L E210K L227G; 270) V2Y N33K F51V L69R K98E V176LH198N E210K L227G; 271) V2Y N33K F51V L69R K98E V176L H198S E210K L227G;272) V2Y N33K F51V L69R K98E V176L H198Y E210K L227G; 273) V2Y N33K F51VL69R K98E V176L E210K S224I L227G; 274) V2Y N33K F51V L69R K98E V176LE210K L227D; 275) V2Y N33K F51V L69R K98E V176L E210K L227G V228P; 276)V2Y N33K F51V L69R K98E V176L E210K L227G I255G; 277) V2Y N33K F51VT143A; 278) V2Y N33K F51V A150G; 279) V2Y N33K F51V H198I; 280) V2Y N33KF51V H198L; 281) V2Y N33K F51V H198N; 282) V2Y N33K F51V H198S; 283) V2YN33K F51V H198Y; 284) V2Y N33K F51V S224I; 285) V2Y N33K F51V L227D;286) V2Y N33K F51V V228P; 287) V2Y N33K F51V I255G; 288) N33K F51I S54TE56K K98I T143A; 289) N33K F51I S54T E56K K98I A150G; 290) N33K F51IS54T E56K K98I H198I; 291) N33K F51I S54T E56K K98I H198L; 292) N33KF51I S54T E56K K98I H198N; 293) N33K F51I S54T E56K K98I H198S; 294)N33K F51I S54T E56K K98I H198Y; 295) N33K F51I S54T E56K K98I S224I;296) N33K F51I S54T E56K K98I L227D; 297) N33K F51I S54T E56K K98IV228P; 298) N33K F51I S54T E56K K98I I255G; 299) N33K F51I E56K L69RK98E T143A; 300) N33K F51I E56K L69R K98E A150G; 301) N33K F51I E56KL69R K98E H198I; 302) N33K F51I E56K L69R K98E H198L; 303) N33K F51IE56K L69R K98E H198N; 304) N33K F51I E56K L69R K98E H198S; 305) N33KF51I E56K L69R K98E H198Y; 306) N33K F51I E56K L69R K98E S224I; 307)N33K F51I E56K L69R K98E L227D; 308) N33K F51I E56K L69R K98E V228P;309) N33K F51I E56K L69R K98E I255G; 310) N33K F51I K98E N101D H198S;311) N33K F51I K98E N101D H198S E210K Y220F A257H; 312) N33K F51I K98EN101D H198S E210K Y220F V228P; 313) N33K F51L S54T E56K K98I T143A; 314)N33K F51L S54T E56K K98I A150G; 315) N33K F51L S54T E56K K98I H198I;316) N33K F51L S54T E56K K98I H198L; 317) N33K F51L S54T E56K K98IH198N; 318) N33K F51L S54T E56K K98I H198S; 319) N33K F51L S54T E56KK98I H198Y; 320) N33K F51L S54T E56K K98I S224I; 321) N33K F51L S54TE56K K98I L227D; 322) N33K F51L S54T E56K K98I V228P; 323) N33K F51LS54T E56K K98I I255G; 324) N33K F51L E56K L69R K98E T143A; 325) N33KF51L E56K L69R K98E A150G; 326) N33K F51L E56K L69R K98E H198I; 327)N33K F51L E56K L69R K98E H198L; 328) N33K F51L E56K L69R K98E H198N;329) N33K F51L E56K L69R K98E H198S; 330) N33K F51L E56K L69R K98EH198Y; 331) N33K F51L E56K L69R K98E S224I; 332) N33K F51L E56K L69RK98E L227D; 333) N33K F51L E56K L69R K98E V228P; 334) N33K F51L E56KL69R K98E I255G; 335) N33K F51L K98E N101D H198S; 336) N33K F51L K98EN101D H198S E210K Y220F V228P; 337) N33K F51L K98E N101D H198S E210KY220F A257H; 338) N33K F51V S54T E56K K98I T143A; 339) N33K F51V S54TE56K K98I A150G; 340) N33K F51V S54T E56K K98I H198I; 341) N33K F51VS54T E56K K98I H198L; 342) N33K F51V S54T E56K K98I H198N; 343) N33KF51V S54T E56K K98I H198S; 344) N33K F51V S54T E56K K98I H198Y; 345)N33K F51V S54T E56K K98I S224I; 346) N33K F51V S54T E56K K98I L227D;347) N33K F51V S54T E56K K98I V228P; 348) N33K F51V S54T E56K K98II255G; 349) N33K F51V E56K L69R K98E T143A; 350) N33K F51V E56K L69RK98E A150G; 351) N33K F51V E56K L69R K98E H198I; 352) N33K F51V E56KL69R K98E H198L; 353) N33K F51V E56K L69R K98E H198N; 354) N33K F51VE56K L69R K98E H198S; 355) N33K F51V E56K L69R K98E H198Y; 356) N33KF51V E56K L69R K98E S224I; 357) N33K F51V E56K L69R K98E L227D; 358)N33K F51V E56K L69R K98E V228P; 359) N33K F51V E56K L69R K98E I255G;360) N33K F51V K98E; 361) N33K F51V K98E N101D H198S; 362) N33K F51VK98E N101D H198S E210K Y220F V228P; 363) N33K F51V K98E N101D H198SE210K Y220F A257H; 364) N33Q G91T P256T; 365) G91T P256T; 366) V230LL259F W260H; 367) L259F W260H; 368) E1C V2K N33K F51I R233C; 369) E1CV2K N33K F51L R233C; 370) E1C V2K N33K F51V R233C; 371) E1C V2Y N33KF51I R233C; 372) E1C V2Y N33K F51L R233C; 373) E1C V2Y N33K F51V R233C;374) V2K N33K F51I L69R K98E V176L E210K L227G; 375) V2K N33K F51I; 376)V2K N33K F51L L69R K98E V176L E210K L227G; 377) V2K N33K F51L; 378) V2KN33K F51V L69R K98E V176L E210K L227G; 379) V2K N33K F51V; 380) V2Y N33KF51I L69R K98E V176L E210K L227G; 381) V2Y N33K F51I; 382) V2Y N33K F51LL69R K98E V176L E210K L227G; 383) V2Y N33K F51L; 384) V2Y N33K F51V L69RK98E V176L E210K L227G; 385) V2Y N33K F51V; 386) N33K F51I S54T E56KK98IF; 387) N33K F51I E56K L69R K98E; 388) N33K F51I K98E N101D G161NH198S E210K Y220F; 389) N33K F51I K98E N101D H198D E210K Y220F; 390)N33K F51I K98E N101D H198S E210K Y220F; 391) N33K F51I K98E N101D H198SE210K; 392) N33K F51I K98E N101D H198S E210R Y220F; 393) N33K F51L S54TE56K K98I; 394) N33K F51L E56K L69R K98E; 395) N33K F51L K98E N101DG161N H198S E210K Y220F; 396) N33K F51L K98E N101D H198D E210K Y220F;397) N33K F51L K98E N101D H198S E210K Y220F; 398) N33K F51L K98E N101DH198S E210K; 399) N33K F51L K98E N101D H198S E210R Y220F; 400) N33K F51VS54T E56K K98I; 401) N33K F51V E56K L69R K98E; 402) N33K F51V K98E N101DG161N H198S E210K Y220F; 403) N33K F51V K98E N101D H198D E210K Y220F;404) N33K F51V K98E N101D H198S E210K Y220F; and 405) N33K F51V K98EN101D H198S E210K; using SEQ ID NO: 2 or SEQ ID NO: 10 for positionnumbering.